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: Frontiers in Immunology

Article Title: Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

doi: 10.3389/fimmu.2019.02920

Figure Lengend Snippet: Characterization of the isolated FcγRIV-specific VHH. (A) The predicted amino acid residue sequence of FcγRIV-VHH-7m, isolated after panning on recombinant mouse FcγRIV protein of a VHH library derived from a llama immunized with immature mouse dendritic cells. Above the sequences the Kabat numbering is indicated. CDR1, −2, and −3 are boxed. (B) Flow cytometric analysis showing binding of FcgRIV-VHH-7m and M2e-VHH-23m to HEK 293T cells transiently transfected with expression vectors coding for mouse FcγRI, FγRIIb, FcγRIII, and FcγRIV along with the common γ-chain for the activating FcγRs and a GFP expression plasmid. The lower graph depicts binding to Mf4/4 cells. The cells were incubated with fourfold serial dilution of Alexa Fluor™ 647 labeled FcγRIV-VHH-7m or M2e-VHH-23m, starting at a concentration of 0.2 μM, binding of the VHHs to the GFP positive cells was analyzed. Data points represent averages of triplicates and error bars standard deviations.

Article Snippet: Human Embryonic Kidney (HEK) 293T cells were transiently transfected with full length mouse FcγRI (MG50086-CF), FcγRIIb (MG50030-CY, SinoBiological Inc.), FcγRIII (MG50326, SinoBiological Inc.) or FcγRIV (MG50036-CF, SinoBiological Inc.) expression constructs along with the common γ-chain for the activating FcγRs (MG50935-CF) by polyethylenimine (PEI)-based transfection.

Techniques: Isolation, Sequencing, Recombinant, Derivative Assay, Binding Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Serial Dilution, Labeling, Concentration 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: 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:

Article Title: CTLA-4 regulates allergen response by modulating GATA-3 protein level per cell

doi: 10.1111/j.1365-2567.2007.02537.x

Figure Lengend Snippet: Effects of in vitro CTLA-4 blockade on GATA-3 protein level/cell and frequency of IL-4-producing cells. Naive CD4+ T cells purified from the spleens of normal mice were induced to differentiate with anti-CD3 mAb, a recombinant form of CD80-Fc, IL-4 and anti-IFN-γ mAb for 3 days. Anti-CTLA-4 mAb was added in soluble form at increasing concentrations (0·1–10 μg/ml). (a) Mean fluorescence intensity for GATA-3 staining, (b) percentage of IL-4+ cells.

Article Snippet: 18 CD4 cell culture For in vitro experiments, splenic naive CD4 cells were purified from non-immunized BALB/c mice by immuno-magnetic cell sorting as previously described 19 and cultured in 24-well plates precoated with anti-CD3ε mAb (clone 145-2C11; 10 μg/ml) and/or recombinant mouse B7-1(CD80)/Fc chimeric protein (R & D Systems, cat. 740-B1; 0·33 μg/ml).

Techniques: In Vitro, Purification, Recombinant, Fluorescence, Staining