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: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Production and characterization of recombinant NSDV GP38-his/FLAG. ( A ) Alignment of partial GPC sequences from different NSDV strains (Ganjam virus IG 619 = NSDV_India; NSDV 708 = NSDV_Kenya; NSDV H. longicornis China = NSDV_China) and CCHFV (IbAr10200). Numbering corresponds to NSDV_India GPC. Amino acid sequences are displayed starting from their respective N-terminus. For CCHFV, the furin protease cleavage site (RSKR) and SKI-1/S1P cleavage site (RRLL) are underlined in blue. For NSDV, arginine-containing motifs reported to be conserved across the GPCs of different orthonairoviruses are highlighted in orange. Two potential N-glycosylation sites (Asn 220 and Asn 400 ) are highlighted with an orange star. ( B ) For protein purification, the partial NSDV_India GPC sequence (aa 138–445; NSDV GP38-his/FLAG protein) was fused to a C-terminal 6×His- and FLAG-tag both highlighted with an orange underline. ( C ) SDS-PAGE of recombinant NSDV GP38-his/FLAG his-tag purified from Sf9 cells followed by Coomassie blue staining and immunoblot analysis using anti-FLAG primary and horseradish peroxidase-conjugated secondary antibodies.

Article Snippet: NSDV Gn ( NAC-REC31904-500 ) and NSDV Gc ( NAC-REC31906-500 ) from The Native Antigen Company were diluted in 0.01 M PBS and coated overnight on Maxisorp 96-well plates at a concentration of 100 ng/well.

Techniques: Recombinant, Virus, Glycoproteomics, Protein Purification, Sequencing, FLAG-tag, SDS Page, Purification, Staining, Western Blot

Journal: Frontiers in Immunology

Article Title: Serological cross-reactivity between Crimean-Congo haemorrhagic fever virus and Nairobi sheep disease virus glycoprotein C

doi: 10.3389/fimmu.2024.1423474

Figure Lengend Snippet: Orthonairovirus test antigens employed during experiments.

Article Snippet: CCHFV Gc , NP_950235 (IbAr10200, Nigeria) , HEK293 (Human) , The Native Antigen Company, UK #REC31696.

Techniques: Expressing, Plasmid Preparation

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Production and characterization of recombinant NSDV GP38-his/FLAG. ( A ) Alignment of partial GPC sequences from different NSDV strains (Ganjam virus IG 619 = NSDV_India; NSDV 708 = NSDV_Kenya; NSDV H. longicornis China = NSDV_China) and CCHFV (IbAr10200). Numbering corresponds to NSDV_India GPC. Amino acid sequences are displayed starting from their respective N-terminus. For CCHFV, the furin protease cleavage site (RSKR) and SKI-1/S1P cleavage site (RRLL) are underlined in blue. For NSDV, arginine-containing motifs reported to be conserved across the GPCs of different orthonairoviruses are highlighted in orange. Two potential N-glycosylation sites (Asn 220 and Asn 400 ) are highlighted with an orange star. ( B ) For protein purification, the partial NSDV_India GPC sequence (aa 138–445; NSDV GP38-his/FLAG protein) was fused to a C-terminal 6×His- and FLAG-tag both highlighted with an orange underline. ( C ) SDS-PAGE of recombinant NSDV GP38-his/FLAG his-tag purified from Sf9 cells followed by Coomassie blue staining and immunoblot analysis using anti-FLAG primary and horseradish peroxidase-conjugated secondary antibodies.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Recombinant, Virus, Glycoproteomics, Protein Purification, Sequencing, FLAG-tag, SDS Page, Purification, Staining, Western Blot

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Seroconversion of NSDV- infected sheep against different NSDV antigens. ( A ) Sera from NSDV-infected sheep ( n = 6) collected at different days post infection (dpi) were tested in duplicate in an indirect in-house ELISA based on recombinant NSDV GP38-his/FLAG. Mean of corrected OD values and standard deviations are displayed. ( B ) Serum samples collected from NSDV-infected sheep ( n = 6) at 0 and 28 dpi were tested for reactivity in an indirect ELISA based on NSDV N-his/FLAG, commercially available recombinant Gn and Gc proteins, and GP38-his/FLAG.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Recombinant, Indirect ELISA

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Immunoblot analysis of NSDV protein expression in SW13 cells. (A) Immunoblot analysis of NSDV-infected and mock-infected SW13 cells collected at 28 h p.i. For detection, newly generated monoclonal antibody (mAb) 6F6 A3B raised against NSDV GP38-his/FLAG and goat anti-mouse IRDye 800-conjugated antibodies were used. The detection of GAPDH served as a loading control. (B) Cell lysates from multi-cycle infection kinetics were collected at the indicated time post infection (p.i.). For detection, mAb 6F6 A3B and mAb 5H11 C1 (raised against NSDV Gc) and rabbit-derived polyclonal serum (R8253) for nucleoprotein (N) detection were used as primary antibodies followed by incubation with goat anti-mouse or anti-rabbit IRDye 680 or 800CW-conjugated secondary antibodies, respectively. β-Tubulin and GAPDH served as loading controls. Representative blots from three independent experiments performed in duplicates are shown.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Western Blot, Expressing, Infection, Generated, Control, Derivative Assay, Incubation

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: NSDV GP38 expression in the supernatant of transfected or NSDV-infected cells. HEK 293T and SW13 cells were transfected with pcDNA NSDV MLD-GP38-Strep (−) or co-transfected with pcDNA NSDV MLD-GP38-Strep and pIR-hfurin encoding for human furin protease (+). Supernatants were harvested at 72 h post-transfection and analyzed by immunoblot using mAb 6F6 A3B. For comparison of NSDV GPC cleavage products, supernatant from NSDV-infected SW13 cells (24 h p.i.) was also analyzed by immunoblot. A representative blot from at least three independent experiments is shown.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Expressing, Transfection, Infection, Western Blot, Comparison

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Efficiency of cleavage of FRET substrates by furin. ( A ) FRET substrates spanning the P7-P4′ amino acid sequences of the putative cleavage site motifs containing an N-terminal o-aminobenzoyl fluorophore and a C-terminal Tyr(3 NO 2 )-NH 2 as a quenching residue were synthesized and tested in an enzyme assay with recombinant furin. Three NSDV GPC sequences were tested: GPC-1 covering the monobasic motif at position 175; GPC-2, a negative control for GPC-1 with the arginine of the motif replaced by alanine, and GPC-3 covering another putative dibasic furin motif for cleavage after residue 134. A FRET substrate with a multibasic furin cleavage site motif of hemagglutinin (HA5) of a highly pathogenic avian influenza virus (HPAIV) strain served as positive control (PC). ( B ) The cleavage of the FRET substrates by recombinant furin was measured in an enzyme kinetic assay. Mean values + SD based on three independent measurements with three independent weights of substrates are displayed.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Residue, Synthesized, Enzymatic Assay, Recombinant, Negative Control, Virus, Positive Control, Kinetic Assay

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Impact of furin inhibition on NSDV glycoprotein processing. SW13 cells were infected with NSDV (MOI of 0.1). After removal of inoculum, fresh medium with (+) or without (−) furin inhibitor MI-1148 (30 µM in DMSO) or DMSO alone was added. At 24 h p.i., cell lysates ( A ) and supernatants ( B ) were collected and analyzed for N, Gc, and GP38 expression in immunoblot. β-Tubulin and GAPDH served as loading controls. Representative blots from three independent experiments each performed in duplicate are shown. ( C ) The signal intensities of the GP38 bands were quantified using Li-Cor software Image Studio Lite Ver 5.2. The GP38 signal intensities from inhibitor-treated samples were set in relation to the signal intensities of the untreated samples (=100%). Mean relative signal intensities and standard deviations from three independent experiments each performed in duplicate are shown.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Inhibition, Infection, Expressing, Western Blot, Software