Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Sialidase protocol validation. All lectins were directly conjugated with Alexa dyes. They were incubated with cells at 1–5 µg/mL for 15 min before a quick wash and cytometry measurement. Compared to untreated control (left), sialidase treatment (right) decreased SNA lectin binding to α2,6 sialylated structures by 15-fold and increased ECL binding to desialylated lactosamine chains (Galβ1,4GlcNAcβ) by an order of magnitude. ( B ) Pseudovirus assay. DsRed fluorescence in HEK293T and stable 293T/ACE2 cells upon addition of VSVG, Spike-WT and Spike-mutant pseudotyped virus. ( C ) Sialidase treatment of pseudovirus. % DsRed positive cell data are shown for study in (main manuscript). Viral entry was sialidase independent. ( D ) Sialidase treatment of HEK/ACE2 cells. Pseudovirus expressing VSVG, Spike-WT and Spike-mutant were added to cells under conditions described in (main manuscript). All error bars are standard deviations. Data are representative of 3 independent runs.

Article Snippet: Antibody , Anti-human F(ab’)two conjugated with Alexa Fluor FITC (goat polyclonal) , Jackson ImmunoResearch , Cat# 109-096-098 RRID: AB_2337668 , Binding assay (1:150).

Techniques: Biomarker Discovery, Incubation, Cytometry, Control, Binding Assay, Fluorescence, Mutagenesis, Virus, Expressing

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: A panel of lectins (from Vector Labs) was conjugated with Alexa dyes, either Alexa 405, 488 or 647. The binding of these fluorescent reagents to wild-type 293T, [N] - 293T and [O] - 293 T cells was measured using flow cytometry. The lectins bound: ( A ) N-glycan high-mannose and complex structures [ConA and LCA bind αMan in high-mannose glycans; PHA-L and PHA-E bind complex glycans], ( B ) lactosamine chains primarily on N-linked glycans [RCA, ECL bind terminal Gal or lactose; DSL bind β1,4GlcNAc], and ( C ) O-glycan related structures [PNA binds Galβ1,GalNAc; VVA and SBA bind GalNAcα]. Measurements were made with either untreated or sialidase treated 293 T cells. As seen: i. Knocking out MGAT1 in [N] - 293T reduces lectin binding in panels A and B (see arrow). ii. Knocking out C1GalT1 results in a dramatic decrease in PNA binding and increase in VVA and SBA binding, These data are consistent with the expected changes in lectin profile upon knocking out these N- and O-glycan-specific enzymes.

Article Snippet: Antibody , Anti-human F(ab’)two conjugated with Alexa Fluor FITC (goat polyclonal) , Jackson ImmunoResearch , Cat# 109-096-098 RRID: AB_2337668 , Binding assay (1:150).

Techniques: Plasmid Preparation, Binding Assay, Flow Cytometry, Glycoproteomics

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human F(ab’)two conjugated with Alexa Fluor FITC (goat polyclonal) , Jackson ImmunoResearch , Cat# 109-096-098 RRID: AB_2337668 , Binding assay (1:150).

Techniques: Binding Assay, Plasmid Preparation, Recombinant, Knock-Out, Derivative Assay

Journal: mAbs

Article Title: Development and characterization of AD-214, an anti-CXCR4 i-body-Fc fusion for the treatment of idiopathic pulmonary fibrosis

doi: 10.1080/19420862.2025.2505090

Figure Lengend Snippet: Construction and characterisation of AD-214. (a) Schematic of AD-214 (left panel). Two molecules of anti-CXCR4 i-body AD-114 (red) fused to an Fc moiety harbouring the DAPA mutation to remove effector function (grey). Western blot of purified AD-214 reduced (left lane) and non-reduced (right lane) probed with anti-Fc antibody (right panel). (b) Chromatogram of AD-214 analysed by size exclusion chromatography (SEC). The retention time for AD-214 was 14.303 min. (c) Sensorgram of AD-214 binding to immobilized human CXCR4. Injected concentrations were 0–20 nM. Binding responses (red line) are overlaid with fit of a simple 1:1 kinetic interaction model (black lines). (d) Sensorgram of human FcRn binding to AD214. Injected concentrations were 0–250 nM. Sensorgrams are shown as coloured lines. (e) AD-214 was screened for specificity using integral molecular’s membrane proteome array, consisting of ∼5,300 human membrane proteins in their native state in unfixed cells. Antibody binding was detected by flow cytometry, and hits were defined as a binding signal more than 3 standard deviations higher than background and validated in follow-up assays. Unlabelled black dots represent hits that were below the defined threshold or that did not validate on retesting. (f) AD-214 binding to human (h)CXCR4 expressed on CHO cells. Bound AD-214 was detected using fluorescently conjugated anti-Fc antibody. MFI, median fluorescence intensity.

Article Snippet: After incubation for 1 h at 4°C, antibodies were removed and cells were washed twice and blocked using anti-human Fc block (Miltenyi, 130-059-901, 1:50) for 15 min at 4°C followed by staining with Alexa FluorTM 647-conjugated goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody (Invitrogen, A-21445) or FITC-conjugated F(ab’)2 Fragment Rabbit Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch, 309-096-008).

Techniques: Mutagenesis, Western Blot, Purification, Size-exclusion Chromatography, Binding Assay, Injection, Membrane, Flow Cytometry, Fluorescence