Journal: mAbs

Article Title: Multivalent IgM scaffold enhances the therapeutic potential of variant-agnostic ACE2 decoys against SARS-CoV-2

doi: 10.1080/19420862.2023.2212415

Figure Lengend Snippet: Avidity-driven binding of ACE2 decoys to SARS-CoV-2 Wuhan (WT) strain spike RBD increases with higher ACE2 valency.

Article Snippet: His-tagged spike RBD proteins for Wuhan (WT) (#10500-CV, R&D Systems), Delta (B.1.617.2) (#10876-CV, R&D Systems), Omicron (B.1.1.529) (#40592-V08H121, Sino Biological), Alpha (B.1.1.7) (#10730-CV, R&D Systems), Beta (B.1.351) (#10735-CV, R&D Systems), and Gamma (P.1) (#10775-CV, R&D Systems) were each covalently immobilized on a CM5 Series S sensor chip by amine coupling to ligand density of ~ 100 RU.

Techniques: Binding Assay

Journal: mAbs

Article Title: Multivalent IgM scaffold enhances the therapeutic potential of variant-agnostic ACE2 decoys against SARS-CoV-2

doi: 10.1080/19420862.2023.2212415

Figure Lengend Snippet: Binding kinetics of ACE2 decoy variants and clinical comparators to recombinant spike protein RBD as measured by SPR.

Article Snippet: His-tagged spike RBD proteins for Wuhan (WT) (#10500-CV, R&D Systems), Delta (B.1.617.2) (#10876-CV, R&D Systems), Omicron (B.1.1.529) (#40592-V08H121, Sino Biological), Alpha (B.1.1.7) (#10730-CV, R&D Systems), Beta (B.1.351) (#10735-CV, R&D Systems), and Gamma (P.1) (#10775-CV, R&D Systems) were each covalently immobilized on a CM5 Series S sensor chip by amine coupling to ligand density of ~ 100 RU.

Techniques: Binding Assay, Recombinant, Control

Journal: Scientific Reports

Article Title: Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana

doi: 10.1038/s41598-020-74904-1

Figure Lengend Snippet: Schematic representation ( a ) and timeline ( b ) for the production of recombinant SARS-CoV-2 RBD and mAb CR3022 in plants by transient gene expression.

Article Snippet: Briefly, 50 μl (2 μg/ml) of the plant-purified SARS-CoV-2 RBD or commercial recombinant CHO-derived SARS-CoV-2 spike RBD-His protein (10534-CV, R & D Systems, USA) was coated on 96-well microplates (Greiner Bio-One GmbH, Germany) and incubated at 4 °C overnight.

Techniques: Recombinant, Expressing

Journal: Scientific Reports

Article Title: Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana

doi: 10.1038/s41598-020-74904-1

Figure Lengend Snippet: Schematic diagram of the T-DNA region of the plant expression vector used in the present study. RB and LB, the right and left borders of the T-DNA region transferred by Agrobacterium into plant cells; P35S: Cauliflower Mosaic Virus (CaMV) 35S promoter, NbPsalK2T1-63 5′UTR: 5′ untranslated region, RBD: SARS-CoV-2 RBD, CR3022 HC: heavy chain of CR3022 antibody, CR3022 LC: light chain of CR3022 antibody, Ext3′FL: 3′ region of tobacco extension gene, Rb7 5′ del: tobacco RB7 promoter, SIR: short intergenic region of BeYDV, LIR: long intergenic region of BeYDV, C2/C1: Bean Yellow Dwarf Virus (BeYDV) ORFs C1 and C2 encoding for replication initiation protein (Rep) and RepA, TMVΩ 5′-UTR: 5′ untranslated region of tobacco mosaic virus Ω, P19: the RNA silencing suppressor from tomato bushy stunt virus; PinII 3′: the terminator from potato proteinase inhibitor II gene.

Article Snippet: Briefly, 50 μl (2 μg/ml) of the plant-purified SARS-CoV-2 RBD or commercial recombinant CHO-derived SARS-CoV-2 spike RBD-His protein (10534-CV, R & D Systems, USA) was coated on 96-well microplates (Greiner Bio-One GmbH, Germany) and incubated at 4 °C overnight.

Techniques: Expressing, Plasmid Preparation, Virus

Journal: Scientific Reports

Article Title: Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana

doi: 10.1038/s41598-020-74904-1

Figure Lengend Snippet: SDS-PAGE and western blot analysis of RBD protein of SARS-CoV-2 produced in N . benthamiana . The crude proteins were extracted from plants, and the RBD protein was purified, analyzed on SDS-PAGE gels and visualized with InstantBlue™ ( a ). Lane M: protein ladder; Lane 1: total soluble protein of N. benthamiana agroinfiltrated with pBY2e-SARS-CoV-2 RBD; Lane 2: purified SARS-CoV-2 RBD. For western blot analysis, proteins on the blot were probed with a rabbit anti-his antibody conjugated with HRP ( b ). Lane 1: crude extract from non-infiltrated N . benthamiana ; Lane 2: total soluble protein of N. benthamiana agroinfiltrated with pBY2e-SARS-CoV-2 RBD; Lane 3: purified SARS-CoV-2-RBD.

Article Snippet: Briefly, 50 μl (2 μg/ml) of the plant-purified SARS-CoV-2 RBD or commercial recombinant CHO-derived SARS-CoV-2 spike RBD-His protein (10534-CV, R & D Systems, USA) was coated on 96-well microplates (Greiner Bio-One GmbH, Germany) and incubated at 4 °C overnight.

Techniques: SDS Page, Western Blot, Produced, Purification

Journal: Scientific Reports

Article Title: Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana

doi: 10.1038/s41598-020-74904-1

Figure Lengend Snippet: Binding of plant-produced RBD to ACE2, the receptor of SARS-CoV-2. Dilutions of plant-produced RBD and commercial CHO-produced RBD (control) were incubated on plates coated with ACE2 and detected with anti-his antibody conjugated with HRP. Non-infiltrated (WT) plant protein was used as a negative control. The data are the mean values of triplicates from each concentration.

Article Snippet: Briefly, 50 μl (2 μg/ml) of the plant-purified SARS-CoV-2 RBD or commercial recombinant CHO-derived SARS-CoV-2 spike RBD-His protein (10534-CV, R & D Systems, USA) was coated on 96-well microplates (Greiner Bio-One GmbH, Germany) and incubated at 4 °C overnight.

Techniques: Binding Assay, Produced, Incubation, Negative Control, Concentration Assay

Journal: Scientific Reports

Article Title: Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana

doi: 10.1038/s41598-020-74904-1

Figure Lengend Snippet: Binding of plant-produced mAb CR3022 to RBD protein. The different concentration of plant-produced mAb CR3022, standard human IgG1, and a plant-produced anti-PD1 antibody (as negative control) were incubated on plates coated either with the plant-produced SARS-CoV-2 RBD ( a ) or a commercial CHO-produced RBD ( c ) and detected with an HRP-conjugated anti-human kappa antibody. In parallel, different dilutions of positive convalescent serum collected from a COVID-19 patient and negative serum were also tested for plant-produced RBD ( b ) and CHO-produced RBD binding ( d ). The data are the mean values of triplicates from each concentration or dilution.

Article Snippet: Briefly, 50 μl (2 μg/ml) of the plant-purified SARS-CoV-2 RBD or commercial recombinant CHO-derived SARS-CoV-2 spike RBD-His protein (10534-CV, R & D Systems, USA) was coated on 96-well microplates (Greiner Bio-One GmbH, Germany) and incubated at 4 °C overnight.

Techniques: Binding Assay, Produced, Concentration Assay, Negative Control, Incubation

Journal: Scientific Reports

Article Title: Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana

doi: 10.1038/s41598-020-74904-1

Figure Lengend Snippet: Specific binding of plant-produced mAb CR3022 to SARS-CoV-2 in infected Vero E6 cells using immunofluorescence. The plant-produced mAb CR3022, positive serum, and a plant-produced anti-PD1 antibody (as negative control) were incubated with SARS-CoV-2-infected and non-infected Vero E6 cells and signal detected with an FITC-conjugated anti-human IgG antibody (green color). Hoechst33342 was used for counterstaining (blue color). The data are the representative images of triplicate assays.

Article Snippet: Briefly, 50 μl (2 μg/ml) of the plant-purified SARS-CoV-2 RBD or commercial recombinant CHO-derived SARS-CoV-2 spike RBD-His protein (10534-CV, R & D Systems, USA) was coated on 96-well microplates (Greiner Bio-One GmbH, Germany) and incubated at 4 °C overnight.

Techniques: Binding Assay, Produced, Infection, Immunofluorescence, Negative Control, Incubation

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: npj Viruses

Article Title: Potent neutralising monoclonal antibodies targeting the spike of NL63 coronavirus

doi: 10.1038/s44298-025-00116-x

Figure Lengend Snippet: a The table depicts plasma IgG endpoint titres against NL63 full length spike and the S1 B domain (RBD), neutralising titres against the Amsterdam1 live virus isolate in HAT24 cells, and frequencies of NL63 spike-specific memory B cells (MBC). Subjects are ranked in order of NL63 spike-specific MBC frequencies. The two subjects that mAbs were isolated from are highlighted in yellow. The ELISA and neutralisation data were from two technical replicates. b Representative flow cytometry plots of NL63 and SARS-CoV-2 spike-specific memory B cells from two subjects stained with recombinant spike proteins fluorescently labelled with PE or APC. c Frequencies of NL63 and SARS-CoV-2 spike-specific memory B cells as a proportion of CD19+ IgD- IgG+ B cells in PBMCs ( N = 14). The line and error bars depict median and interquartile range respectively. Statistical analyses between matched pairs were performed with a Wilcoxon signed-rank test (*** P < 0.001). Results in this figure are representative data from single experiments.

Article Snippet: 96-well Maxisorp plates (Thermo Fisher) were coated overnight at 4 °C with 1.5 µg/mL of recombinant NL63 RBD protein (R&D systems, #10605-CV-100) or 1.5 µg/ml of BSA in PBS.

Techniques: Virus, Isolation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Recombinant

Journal: npj Viruses

Article Title: Potent neutralising monoclonal antibodies targeting the spike of NL63 coronavirus

doi: 10.1038/s44298-025-00116-x

Figure Lengend Snippet: a Organisation of the NL63 spike protein. Upper part: linear depiction of NL63 spike genome (not to scale). The S1 subunit consists of domains 0, A, B, C and D. Lower part: cryo-ET structure of the trimeric NL63 spike (PDB: 8FR7) with the subdomains of one protomer shown in coloured ribbons. The other two protomers are shown in grey. b Germline identity and CDR3 sequences of 9 NL63 spike-specific human mAbs. c Binding and neutralisation specificity of NL63 mAbs. Binding of mAbs was assessed via ELISA against full length spike and subdomains S1 B , S1 0 , S1 0 + S1 A from NL63. Breadth was assessed against spike proteins from the ChinaGD02 NL63 strain, 229E, SADS-CoV (swine acute diarrhoea syndrome coronavirus) and SARS-CoV-2. Receptor binding inhibition was assessed via a S1 B (RBD)-ACE2 inhibition ELISA while neutralisation was assessed using live NL63 virus isolates (Amsterdam1 and contemporary isolates) in HAT24 cells. ELISA binding and neutralisation titration curves are provided in Supplementary Fig. and . Dashes indicate no binding or neutralising activity. The receptor binding inhibition ELISA and neutralisation assays were performed in duplicate and are representative data from single experiments.

Article Snippet: 96-well Maxisorp plates (Thermo Fisher) were coated overnight at 4 °C with 1.5 µg/mL of recombinant NL63 RBD protein (R&D systems, #10605-CV-100) or 1.5 µg/ml of BSA in PBS.

Techniques: Tomography, Binding Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Virus, Titration, Activity Assay

Journal: npj Viruses

Article Title: Potent neutralising monoclonal antibodies targeting the spike of NL63 coronavirus

doi: 10.1038/s44298-025-00116-x

Figure Lengend Snippet: a Antibody escape viruses were generated by serial passaging of NL63 (Amsterdam1 strain) in HAT24 cells in the presence of neutralising mAbs (NLH02, NLH04, NLH07, NLH18). At each passage, the wells with detectable cytopathic effect (CPE) at the highest mAb concentration were harvested and re-passaged in a titration of mAbs starting at a higher concentration. Serial passaging was repeated until escape occurred (presence of CPE) at a mAb concentration of 50 µg/ml. Whole genome sequencing was performed on viral RNA extracted from cell supernatants. Amino acid mutations within spike that become fixed are indicated when they first appear. b mAb escape mutations mapped onto NL63 spike (PDB: 8FR7) with the top view of spike depicted on the left and side view depicted on the right. S1 B (RBD) domains of each spike protomer are coloured. Escape mutations for NLH04, NLH07 and NLH18 are within domain S1 B (RBD) while the escape mutations for NLH02 are within the S2 domain.

Article Snippet: 96-well Maxisorp plates (Thermo Fisher) were coated overnight at 4 °C with 1.5 µg/mL of recombinant NL63 RBD protein (R&D systems, #10605-CV-100) or 1.5 µg/ml of BSA in PBS.

Techniques: Generated, Passaging, Concentration Assay, Titration, Sequencing

Journal: npj Viruses

Article Title: Potent neutralising monoclonal antibodies targeting the spike of NL63 coronavirus

doi: 10.1038/s44298-025-00116-x

Figure Lengend Snippet: A Amino acid conservation across NL63 strains ( N = 207 sequences spanning 1983—2023 from Genbank) is illustrated on a monomer of NL63 spike (PDB: 8FR7). Shading indicates areas of low ( < 80%; deep red), partial ( > 80%; salmon), near complete ( > 0.98%; light pink) and complete conservation (100%; grey). B Amino acid conservation depicted on NL63 domain S1 B (RBD) in complex with the ACE2 receptor (PDB: 3KBH). C Amino acid sequence conservation of the residues around the escape mutations (in black boxes) for the S2 neutralising mAb (NLH02) and three RBD neutralising mAbs (NLH04, NLH07 and NLH18). Residues are shaded according to how well conserved they are with orange/red indicating lower conservation.

Article Snippet: 96-well Maxisorp plates (Thermo Fisher) were coated overnight at 4 °C with 1.5 µg/mL of recombinant NL63 RBD protein (R&D systems, #10605-CV-100) or 1.5 µg/ml of BSA in PBS.

Techniques: Sequencing

Journal: bioRxiv

Article Title: A synthetic delivery vector for mucosal vaccination

doi: 10.1101/2023.05.15.540756

Figure Lengend Snippet: sSTxB(70C)-RBD induced higher RBD-specific CD8 + T cell and mucosal IgA responses in the lung than non-vectorized RBD. a) Conjugation of RBD to sSTxB(70C) through thiol-maleimide reaction. b-d) Balb/c mice (3-4 per group) were immunized at D0 and D14 via the intranasal route with 0.5 nmol of sSTxB(70C)-RBD or RBD, both combined with c-di-GMP as an adjuvant. Mice were sacrificed on day 21. Naive non-immunized mice were used as controls. After perfusion, the lungs were collected, and cells dissociated. CD8 + T cells were then purified and submitted to an IFNγ Elispot assay in which cells were sensitized or not (medium) with the S1 peptide pool encompassing the RBD sequence of SARS-CoV-2 (b). Bronchoalveolar lavage was also collected at day 21, diluted to ½, upon which specific anti-RBD IgA (c) and IgG (d) were measured. One out of 3 representative independent experiments is shown. Mean±SEM Mann-Whitney t-test. *P < 0.05.

Article Snippet: Recombinant SARS-CoV-2 spike RBD protein (R&D Biotechne,#10534-CV-100) in sodium carbonate buffer pH 9.6 was coated at 2 μg/mL (75 μL/well) overnight at 4 °C onto 96-well Maxisorp clear plates.

Techniques: Conjugation Assay, Purification, Enzyme-linked Immunospot, Sequencing, MANN-WHITNEY

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