Journal: bioRxiv

Article Title: Physical, chemical, and structural properties of human gastric organoid-derived mucus

doi: 10.1101/2025.11.08.687257

Figure Lengend Snippet: ( A ) Alcian blue staining for acidic mucins on tissue surface epithelium (top) and in the organoid lumen (bottom). Representative of 3 experiments. ( B ) Immunofluorescence staining for MUC5AC (green) on the surface epithelium and in the pit regions of human gastric tissue (top) and in the organoid lumen (bottom). Representative of two experiments. ( C ) Immunofluorescence staining for MUC6 in the neck regions of the gastric glands and on the outer edges of the organoid lumen. Representative of 2 experiments. All scale bars = 200 μm.

Article Snippet: Sections were blocked for 30 min with animal-free blocking reagent (Vector Laboratories) and incubated with primary antibodies to E-cadherin (Biolegend Cat. 324102), MUC5AC (Santa Cruz Biotech Cat. 398985), and MUC6 (Invitrogen Cat. PA5-50557) overnight at 4°C.

Techniques: Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Physical, chemical, and structural properties of human gastric organoid-derived mucus

doi: 10.1101/2025.11.08.687257

Figure Lengend Snippet: (A ) Timeline for 2D culture conditions, mucus collection, and functional analyses. ( B ) Time-dependent development of transepithelial electrical resistance (TEER); one representative out of seven experiments with four technical replicates. Mean ± SD; dotted line indicates TEER threshold for airlift at y=200 Ωcm 2 . ( C ) Schematic diagram of Transwell 2D culture conditions: submerged (left) and air-liquid interface, ALI (right). ( D ) Clear, viscoelastic mucus on the Transwell insert (left) and removed from the apical epithelium (right). ( E ) Bioengineered mucus (BGM) dry mass collected per well every other day (mean ± SD), representative of two experiments with 3-5 technical replicates. Dry mass was determined by weighing lyophilized samples. ( F ) Concentration (mg dry weight per mL) of BGM (n=23), native mucus (n=6), and L-WRN culture media (n=8). Data analyzed by one-way ANOVA with Tukey’s multiple comparisons test; **** P ≤ 0.0001. ( G ) Concentration of MUC5AC in BGM (n=10) and NM (n=5) determined by ELISA. ( H ) Size exclusion chromatography paired with multi-angle light-scattering (SEC-MALS) analysis of BGM (top) and NM (bottom) showing the presence of low and high molecular weight compounds based on differential refractive indices (dRI) in blue and the light scattering (LS) signal in red. ( I ) Molecular weights of a porcine gastric mucin (PGM) control, BGM (n=3), and NM (n=4) determined by SEC-MALS. Data analyzed by one-way ANOVA with Tukey’s multiple comparisons test. All bar graphs include individual data points, mean ± SD.

Article Snippet: Sections were blocked for 30 min with animal-free blocking reagent (Vector Laboratories) and incubated with primary antibodies to E-cadherin (Biolegend Cat. 324102), MUC5AC (Santa Cruz Biotech Cat. 398985), and MUC6 (Invitrogen Cat. PA5-50557) overnight at 4°C.

Techniques: Functional Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Size-exclusion Chromatography, Multi-Angle Light Scattering, High Molecular Weight, Control

Journal: bioRxiv

Article Title: Physical, chemical, and structural properties of human gastric organoid-derived mucus

doi: 10.1101/2025.11.08.687257

Figure Lengend Snippet: Three biological replicates of BGM, four native human gastric mucus samples collected from surgical material, and one porcine gastric mucin sample were analyzed by LC/MC. ( A ) Number of proteins identified by mass spectrometry in bioengineered gastric mucus (BGM; n=3), native mucus (NM; n=4), and porcine gastric mucin (PGM; n=1) samples. ( B ) Number of total and shared proteins (overlapping region) between BGM and NM (top) and BGM and PGM (bottom). ( C ) Relative abundance of gastrointestinal mucins MUC5AC, 1, 6, and 2 and trefoil factors (TFF) 1 and 2 in BGM (blue), NM (red), and PGM (grey). Combined data from all identified protein isotypes. ( D ) Categorization of proteins detected in PGM (left) and BGM (right) based on protein functions and cellular distribution listed in the Human Protein Atlas . Percentage values represent cumulative relative protein intensity values for each category. ( E ) Heatmap showing relative expression of key stomach-specific factors identified in BGM and NM. Data from n=4 NM and n=3 BGM samples.

Article Snippet: Sections were blocked for 30 min with animal-free blocking reagent (Vector Laboratories) and incubated with primary antibodies to E-cadherin (Biolegend Cat. 324102), MUC5AC (Santa Cruz Biotech Cat. 398985), and MUC6 (Invitrogen Cat. PA5-50557) overnight at 4°C.

Techniques: Mass Spectrometry, Expressing

Journal: bioRxiv

Article Title: Selenoprotein P Deficiency Drives Hepatocellular Carcinoma Progression via Induction of Neutrophil Senescence and Immunosuppressive Microenvironment

doi: 10.1101/2025.06.24.661430

Figure Lengend Snippet: (a) Flow cytometric analysis showing an increased percentage of neutrophils (CD11b + lymphocyte antigen 6 family member G-positive (Ly6G + ) cells) in the tumor microenvironment of sh Sepp1 mice compared to shNC controls (n = 5–6 per group). (b) Quantitative RT-PCR analysis of senescence-associated markers Cdkn1a and Cdkn2a in tumor-infiltrating neutrophils (TINs) (n = 5–6 per group). (c) mRNA expression levels of S100a8 and S100a9 were significantly elevated in TINs from sh Sepp1 mice compared to shNC controls (n = 6 per group). (d) mRNA expression levels of Chil3 , Cd14 , and Vegfa were significantly elevated in TINs from sh Sepp1 mice compared to shNC controls (n = 5-6 per group). (e) Flow cytometric analysis of S100A8/9 protein expression in neutrophils, with representative histogram overlay (left) and quantification of mean fluorescence intensity (MFI) (right) (n = 3 per group). (f) Flow cytometric quantification of CD3 + T cells in the tumor microenvironment of shNC and sh Sepp1 mice, indicating significantly decreased T cell infiltration in sh Sepp1 tumors compared to controls (n = 6 per group). (g–i) Functional analysis of CD8 + T cells in tumors, demonstrating increased programmed cell death 1 (PD-1) expression (g), decreased granzyme B (GZMB) levels (h), and decreased interferon gamma (IFNγ) levels (i) in sh Sepp1 mice compared to controls (n = 5–6 per group). (j) The percentage of natural killer (NK) cells (NK1.1 + CD3 − ) was significantly decreased in the tumor microenvironment of sh Sepp1 mice compared to shNC animals (n = 6 per group). (k) Schematic representation of the experimental setup for assessing T cell suppression by TINs. Neutrophils were isolated from the tumors of mice injected with shNC or sh Sepp1 constructs using Magnetic Activated Cell Sorting (MACS). These neutrophils were co-cultured with CFSE-labeled OT-1 splenocytes from OT-1 mice at various ratios (2:1 to 1:8) in the presence of OVA 257−264 peptide for 48 hours, followed by analysis using FACS and ELISA. (l) T cell proliferation analysis measuring the percentage of proliferating OT-1 T cells co-cultured with neutrophils isolated from shNC or sh Sepp1 tumors. This analysis showed significantly decreased T cell proliferation in the presence of neutrophils from Sepp1 -depleted tumors across different neutrophil: OT-1 ratios (compared to those cultured in the presence of neutrophils from shNC tumors) (n = 3 per group). (m) Representative flow cytometry histograms of CFSE dilution in OT-1 splenocytes, illustrating T cell proliferation at different neutrophil: OT-1 splenocyte ratios. Data are presented as mean ± SEM; statistical significance was determined using two-tailed unpaired Student’s t-tests.

Article Snippet: For assessments of spent medium samples, ELISA was performed using IFNγ (Multi Sciences, Cat. EK280) and IL2 (Multi Sciences, Cat. EK202) ELISA kits according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Fluorescence, Functional Assay, Isolation, Injection, Construct, FACS, Cell Culture, Labeling, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Selenoprotein P Deficiency Drives Hepatocellular Carcinoma Progression via Induction of Neutrophil Senescence and Immunosuppressive Microenvironment

doi: 10.1101/2025.06.24.661430

Figure Lengend Snippet: (a) Flow cytometric analysis of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) from shNC and sh Sepp1 mice. (b, c) Relative mRNA levels of Cdkn2b (b) and Cdkn2d (c) in neutrophils isolated from shNC and sh Sepp1 animals (n=5-6 per groups). (d) Flow cytometric analysis of T cell proportions in shNC and sh Sepp1 mice. (e-f) CD4 + T cells and CD8 + T cells proportions in the tumor microenvironment of shNC and sh Sepp1 mice. (g) Programmed cell death protein 1 (PD-1) expression in CD8 + T cells, as assessed by flow cytometry. (h) Cytokine staining of intracellular interferon gamma (IFNγ) in CD8 + T cells from shNC and sh Sepp1 mice. (i, j) ELISA quantification of IFNγ (d) and IL-2 (e) in the co-culture spent medium, demonstrating significantly decreased levels of both of these cytokines in cultures employing neutrophils isolated from Sepp1 -depeleted tumors (compared to neutrophils isolated from control tumors) across various neutrophils: OT-1 splenocyte ratios (n = 3 per group). Data are presented as mean ± SEM; statistical significance was determined using two-tailed unpaired Student’s t-tests.

Article Snippet: For assessments of spent medium samples, ELISA was performed using IFNγ (Multi Sciences, Cat. EK280) and IL2 (Multi Sciences, Cat. EK202) ELISA kits according to the manufacturer’s instructions.

Techniques: Derivative Assay, Isolation, Expressing, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Control, Two Tailed Test

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function.

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: Fig. 2. α2B/2C agonism suppresses chronic corneal epitheliopathy in dry eye disease (DED) and restores immunohomeostasis. (A) Treatment with the α2B/ 2C agonist (AGN-762) after the development of chronic DED effectively ameliorates the disease, assessed by corneal fluorescein staining (CFS) score. ****, p < 0.0001 vs untreated or vehicle group; n = 30 eyes (15 mice) /group from two independent experiments. (B) Corneal inflammation was assessed at the end of treatment by quantifying mature MHC-II+CD11b+ cells using flow cytometry and protein levels of IL-1β and IL-6 using ELISA. The upper panel shows the gating strategy for flow cytometry analysis. (C) Conjunctival inflammation was assessed by quantifying infiltrating CD4+ T cells using flow cytometry and protein levels of IL-17A, IL-17F, and IFN-γ using ELISA. The upper panel of the flow plots shows the gating strategy for flow cytometry analysis. (D) Draining lymph node (DLN) Th17 response was determined by flow cytometry. The upper panel shows the gating strategy for flow cytometry analysis. (E) The suppressive function of Treg cells on cell proliferation was quantified using the BrdU incorporation assay and compared with the proliferative responses in the absence of Treg (0 % suppression). For flow cytometry of corneal and conjunctival tissues, samples were pooled from 5 eyes in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; data shown were from one representative experiment out of two performed and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: In vitro Th17 cell polarization Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function.

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: Fig. 3. α2B/2C agonism provides sustained amelioration of corneal epitheliopathy and suppresses disease exacerbation. (A) A 7-day treatment with the α2B/2C agonist (AGN-762) in chronic DED (day 21 – 28) effectively maintains therapeutic efficacy for months (day 28 – 56), and suppresses disease exacerbation in treated animals upon re-challenged with desiccating stress (day 56 – 63). Disease severity was assessed by corneal fluorescein staining (CFS) score. ***, p < 0.001; ****, p < 0.0001 vs untreated or vehicle group; n = 20 eyes (10 mice) /group from two independent experiments. (B) Corneal inflammation was assessed at day 63 by quantifying mature MHC-II+CD11b+ cells using flow cytometry and protein levels of IL-1β and IL-6 using ELISA. The same gating strategy for flow cytometry analysis was used as shown in Fig. 2B. (C) Conjunctival inflammation was assessed at day 63 by quantifying protein levels of IL-17A, IL-17F, and IFN-γ using ELISA. (D) Draining lymph node Th17 response was determined by flow cytometry at day 63. The same gating strategy for flow cytometry analysis was used as shown in Fig. 2D. (E) The suppressive function of Treg cells on cell proliferation was quantified using the BrdU incorporation assay and compared with the proliferative responses in the absence of Treg (0 % suppression). For flow cytometry of corneal and conjunctival tissues, samples were pooled from 5 eyes in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant; data shown were from one representative experiment out of two performed and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: In vitro Th17 cell polarization Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Drug discovery, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function.

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: Fig. 5. α2B/2C agonism (by AGN-762 at the higher concentration) enhances DED-derived effector T cells (Teff) in vitro. (A) Flow cytometry plots show the purity of freshly sorted Teff cells defined as CD4+CD25-Foxp3- cells that were subjected to cell cultures in vitro. (B) Flow cytometric analysis of CD4+CD25- Teff after 24 h culturing with AGN-762, with representative dot plots shown (the upper panel shows the gating strategy), and frequencies of IL-17A+IFN-γ- Th17, IL-17A+IFN- γ+ Th17/1, and IL-17A-IFN-γ+ Th1 summarized in the bar graphs. (C) ELISA assay on the culture supernatants for the levels of IL-17A and IFN-γ. Data shown were

Article Snippet: In vitro Th17 cell polarization Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Derivative Assay, In Vitro, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function.

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: Fig. 6. Adoptive transfer of ex vivo AGN-762-treated DED-Treg effectively reduces corneal epitheliopathy severity and Th17 immunity. (A) Treg cells were isolated from DED mice and in vitro cultured with 1 μM AGN-762 or vehicle for 24 h. The freshly sorted effector CD4+ T cells from DED (Teff) along with normal Treg, DED-Treg pre-treated with the vehicle, or DED-Treg pre-treated with AGN-762 were adoptively transferred to Rag1-/- mice, which were subsequently exposed to desiccating stress for 6 days. Disease severity was assessed by corneal fluorescein staining (CFS) score, and summarized from two independent experiments. n = 10–14 eyes (5–7 mice) /group. (B) Flow cytometric analysis of T cell response in the draining lymph nodes of Rag1-/- recipients at day 6, with representative dot plots shown on the left (the upper panel shows the gating strategy) and frequencies of IL-17-producing CD4 + T cells (including both IL-17A+IFN-γ- Th17 and IL-17A+IFN- γ+ Th17/1) summarized from two independent experiments on the right. *, p < 0.05; **, p < 0.01; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: In vitro Th17 cell polarization Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Adoptive Transfer Assay, Ex Vivo, Isolation, In Vitro, Cell Culture, Staining

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function.

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: Fig. 7. Neutralization of IL-10 abrogates AGN-762-mediated restoration of Treg immunosuppressive function. (A) DED-Treg cells were pre-treated with 1 μM AGN-762 or vehicle for 24 h and then subjected to suppressive function assay in the presence of anti-IL-10 neutralizing antibody or isotype IgG control. (B) Treg cells were isolated from DED mice and in vitro cultured with 1 μM AGN-762 for 24 h. The freshly sorted effector CD4+ T cells from DED (Teff) along with DED-Treg pre- treated with AGN-762 were adoptively transferred to Rag1-/- mice, which were subsequently exposed to desiccating stress for 6 days. The recipients received treatment with anti-IL-10 antibody or control IgG immediately after adoptive transfer (day 0) as well as at day 3. Disease severity was assessed by corneal fluorescein staining (CFS) score, and summarized from two independent experiments. n = 16 eyes (8 mice) /group (C) Flow cytometric analysis of T cell response in the draining lymph nodes of Rag1-/- recipients at day 6, with representative dot plots shown on the left and frequencies of total T cells and IL-17-producing CD4 + T cells (including both IL-17A+IFN-γ- Th17 and IL-17A+IFN-γ+ Th17/1) summarized from two independent experiments on the right. *, p < 0.05; **, p < 0.01; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test (A) or unpaired t test (B and C).

Article Snippet: In vitro Th17 cell polarization Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Neutralization, Functional Assay, Control, Isolation, In Vitro, Cell Culture, Adoptive Transfer Assay, Staining

Journal: Antiviral research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein.

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Fig. 1. Structural elements in the ACE2-Fc constructs. a Schematic depiction of the main parts in an engineered ACE2-Fc molecule and their functional properties. b Design of the ACE2-Fc fusion protein; ACE2 parts in light and dark blue, IgG-Fc part in gray, spike (S) protein trimer in green and the receptor-binding domain (RBD) located at the tip of each spike protein in orange and dark green. The binding region as well as active site residues H374 and H378 important for the enzymatic activity of ACE2 are highlighted. Structures of the following Protein Data Bank (PBD) identifiers were used for modeling: 6M17, 6M0J, 6VSB, 5DK3. c Nomenclature and structural variations in the ACE2-Fc constructs.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 ◦C and 5% CO2.

Techniques: Construct, Functional Assay, Binding Assay, Activity Assay

Journal: Antiviral research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein.

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Fig. 2. Structural and functional characteristics of the ACE2-Fc proteins. a Far-UV CD spectra (left) and Near-UV CD spectra (right) of ACE2-Fc constructs indicating that all proteins exhibit similar secondary and tertiary structures. b Chromatograms and molecular mass from size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) indicating that the ACE2-Fc molecules form homodimers. c Non-reducing (top) and reducing (bottom) sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) analysis showing that intermolecular disulfide bonds in the homodimers are formed. d Comparison of fluorescence signals over time obtained in an assay testing the cleavage of a fluorescent peptidyl-4-methylcoumaryl-7-amide (MCA). Relative fluorescent units (RFU) are given. e Amount of MCA cleaved after 30 min of incubation with the ACE2-Fc constructs. Ref1 and Ref2 are two different commercially available ACE2-Fc proteins from Genscript and Acrobiosystems, respectively. Bars are mean values; error bars depict the 95% confidence interval of six independent experiments shown as circles.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 ◦C and 5% CO2.

Techniques: Functional Assay, Circular Dichroism, Construct, Size-exclusion Chromatography, Multi-Angle Light Scattering, SDS Page, Comparison, Fluorescence, Incubation

Journal: Antiviral research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein.

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Fig. 3. Interaction of the ACE2-Fc con structs with the SARS-CoV-2 RBD. a Surface plasmon resonance (SPR) was performed to obtain binding curves of ACE2-Fc fusion constructs and an unre lated Fc fusion protein (aflibercept) to an immobilized RBD from SARS-CoV-2. An exemplary binding curve is shown (RU = Response Units). b Binding con stants of the ACE2-Fc constructs to wards the RBD of SARS-CoV-2 (Mean ± SD of triplicate measurements). c ACE2- Fc fusion proteins were pre-incubated with the SARS-CoV-2 spike S1 protein and tested in a competition ELISA for their ability to neutralize S1 binding to immobilized ACE2 protein. Potent inhi bition of SARS-CoV-2 spike S1 protein by ACE2-IgG4-Fc constructs (left) and ACE2-IgG1-Fc constructs (right). Data are represented as means ± SD of at least two independent experiments.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 ◦C and 5% CO2.

Techniques: SPR Assay, Binding Assay, Construct, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Antiviral research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein.

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Fig. 4. Effect of ACE2-Fc on SARS-CoV-2-GFP infection and inhibition of SARS-CoV-2 primary isolates. a ACE2-IgG4-Fc reduces SARS-CoV-2-GFP replication. Representative fluorescent images of Vero E6 cells infected with SARS-CoV-2-GFP (multiplicity of infection (MOI) = 0.6 IU/cell) pre-incubated with ACE2-IgG4-Fc fusion construct 1 (632 nM). b ACE2-Fc fusion proteins potently neutralize coronaviruses. Serial dilutions of ACE2-Fc fusion proteins were pre-incubated with different coronaviruses and tested for their ability to neutralize the virus before infection of Vero E6 cells. Neutralization of SARS-CoV (top), SARS-CoV-2-Jan (middle) and SARS-CoV-2-April (bottom) by ACE2-IgG4-Fc constructs (left) and ACE2-IgG1-Fc constructs (right) is shown. Data given are means ± SEM of three independent experiments each. 50% inhibitory concentrations (IC50) determined as well as the 95% confidence interval (CI 95%) are given for each construct. The dashed lines indicate the IC50 values on the corresponding curves.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 ◦C and 5% CO2.

Techniques: Infection, Inhibition, Incubation, Construct, Virus, Neutralization

Journal: Antiviral research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein.

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Fig. 5. Neutralization potency of ACE2- IgG4-Fc fusion proteins increases with evolution of pandemic SARS-CoV-2 variants. Serial dilutions of ACE2-IgG4- Fc fusion constructs 1 and 3 were pre- incubated with the indicated SARS- CoV-2 primary isolates or VoCs and tested for their ability to prevent cyto toxicity following infection of A549- hACE2 cells. Neutralization of SARS- CoV-2-Jan, SARS-CoV-2-April and SARS-CoV-2 VoCs alpha, beta and delta by enzymatically active ACE2-IgG4-Fc construct 1 (left) and enzymatically inactive ACE2-IgG4-Fc construct 3 (right). Data given are means ± SEM of three independent experiments each. 50% inhibitory concentrations (IC50 values) determined as well as the 95% confidence interval (CI 95%) are shown for each construct. The dashed lines indicate the IC50 values on the corre sponding curves.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 ◦C and 5% CO2.

Techniques: Neutralization, Construct, Incubation, Infection

Journal: International Journal of Molecular Sciences

Article Title: Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies

doi: 10.3390/ijms23158470

Figure Lengend Snippet: Binding analysis of STRE and CBA biopanning derived phage pools and individual phage clones. ( A ) Polyclonal phage ELISA against the recombinant antigens mβc (mu-sAIC2B-His, positive) and mβ IL-3 (mu-sAIC2A-His, negative). Phage pools from all three CBA (cR1-cR3) and STRE (sR1-sR3) rounds were compared to the naïve Jones-Mahler (JM) library. Binding was assessed in duplicates by immobilizing the recombinant proteins on ELISA plates followed by incubation with successive 10-fold dilutions of phage particles. ( B ) Polyclonal flow cytometry analysis of the original phage library and subsequent biopanning pools against transiently expressed mβc-GFP on the surface of CHO-XL99 cells. Protein expression was monitored using the intracellular GFP ( x -axis), while phage binding was determined using a primary mouse anti-M13 antibody in combination with a secondary anti-mouse IgG antibody conjugated to DyLight650 ( y -axis). ( C ) Monoclonal phage ELISA of selected purified phage clones that exceeded an A 450 reading of 0.6 in a preliminary screen . Adipose fatty acid binding protein (AFBP) was used as a non-specific His-tagged negative control. Prefixes ‘c’ and ‘s’ denote clones derived from CBA and STRE biopanning methods, respectively. Clones with no prefix were identified in both biopanning campaigns.

Article Snippet: PE-conjugated rat anti-mouse CD131 (mβc) antibody (BD Pharmingen, cat# 559920, RRID: AB_397374); Mouse anti-M13 antibody (SinoBiological, Beijing, China; cat# 11973-MM05T, RRID: AB_2857926); HRP conjugated goat anti-mouse IgG antibody (BioRad, South Granville, NSW, Australia; cat# 1706516, RRID: AB_11125547); DyLight 650 conjugated goat anti-mouse IgG antibody (Abcam, Melbourne, VIC, Australia; cat# 96874, RRID:AB_10679531); Isotype mouse IgG2a control antibody (Miltenyi Biotec, Macquarie Park, NSW, Australia; cat# 130-106-546, RRID:AB_2661589); PE-conjugated mouse anti-STAT5 (pY694) antibody (BD Biosciences, Macquarie Park, NSW, Australia; cat# 612567, RRID:AB_399858); PE-conjugated rat anti-mouse CD123 (IL-3Rα) antibody (eBioscience, San Diego, CA, USA; cat# 12-1231-82, RRID:AB_465839); AF488 conjugated rat anti-mouse β IL-3 antibody (R&D Systems, Minneapolis, MN, USA; cat# FAB5492G, RRID: RRID:AB_2905558); Rat anti-mouse CD131 (mβc) antibody (BD Biosciences, cat# 740050, RRID:AB_2739817).

Techniques: Binding Assay, Derivative Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Flow Cytometry, Expressing, Purification, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies

doi: 10.3390/ijms23158470

Figure Lengend Snippet: Analytical size exclusion chromatography (SEC) of purified anti-mβc mAbs. Chromatograms show the elution profile of each mAb (blue) overlayed on a standard run of proteins with known molecular weights ( M W ) (grey). ( A ) All 30 candidates eluted in a single peak indicative of a homogenous species of molecules in solution. However, six antibodies (cA5, cC4, cG8, sC3, sC9, and sE7) exhibited longer retention times than expected for a full-length IgG molecule when analyzed on a silica-based column. ( B ) These clones were further assessed on a Superdex 200 column coupled to a MALS detector for accurate M W determination. Retention times for all antibodies except sC3 were comparable to the IgG protein in the standard (peak B), and M W s corresponded with theoretically calculated values. Standard proteins: A—thyroglobulin (670 kDa), B—IgG (158 kDa), C—ovalbumin (44 kDa), D—myoglobin (17 kDa), E—vitamin E12 (1.35 kDa).

Article Snippet: PE-conjugated rat anti-mouse CD131 (mβc) antibody (BD Pharmingen, cat# 559920, RRID: AB_397374); Mouse anti-M13 antibody (SinoBiological, Beijing, China; cat# 11973-MM05T, RRID: AB_2857926); HRP conjugated goat anti-mouse IgG antibody (BioRad, South Granville, NSW, Australia; cat# 1706516, RRID: AB_11125547); DyLight 650 conjugated goat anti-mouse IgG antibody (Abcam, Melbourne, VIC, Australia; cat# 96874, RRID:AB_10679531); Isotype mouse IgG2a control antibody (Miltenyi Biotec, Macquarie Park, NSW, Australia; cat# 130-106-546, RRID:AB_2661589); PE-conjugated mouse anti-STAT5 (pY694) antibody (BD Biosciences, Macquarie Park, NSW, Australia; cat# 612567, RRID:AB_399858); PE-conjugated rat anti-mouse CD123 (IL-3Rα) antibody (eBioscience, San Diego, CA, USA; cat# 12-1231-82, RRID:AB_465839); AF488 conjugated rat anti-mouse β IL-3 antibody (R&D Systems, Minneapolis, MN, USA; cat# FAB5492G, RRID: RRID:AB_2905558); Rat anti-mouse CD131 (mβc) antibody (BD Biosciences, cat# 740050, RRID:AB_2739817).

Techniques: Size-exclusion Chromatography, Purification, Clone Assay, Multi-Angle Light Scattering

Journal: International Journal of Molecular Sciences

Article Title: Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies

doi: 10.3390/ijms23158470

Figure Lengend Snippet: Flow cytometric assessment of purified mAbs binding to NFS-60 cells (dark grey) natively expressing the mβc receptor. Mouse M1 cells (light grey), which do not express mβc, as well as the PE-conjugated secondary antibody and an unrelated mIgG2a isotype were used as negative controls. Cells were incubated with 200 μg/mL of each mAb, and binding is shown as a measure of the mean fluorescence intensity (MFI). Despite being isolated from the CBA biopanning method, corresponding mAbs (prefix ‘c’) generally produced a less pronounced shift indicative of a lower binding affinity for the target molecule in the native cell surface environment. Prefix ‘s’—STRE biopanning method; no prefix—binders isolated from both methods.

Article Snippet: PE-conjugated rat anti-mouse CD131 (mβc) antibody (BD Pharmingen, cat# 559920, RRID: AB_397374); Mouse anti-M13 antibody (SinoBiological, Beijing, China; cat# 11973-MM05T, RRID: AB_2857926); HRP conjugated goat anti-mouse IgG antibody (BioRad, South Granville, NSW, Australia; cat# 1706516, RRID: AB_11125547); DyLight 650 conjugated goat anti-mouse IgG antibody (Abcam, Melbourne, VIC, Australia; cat# 96874, RRID:AB_10679531); Isotype mouse IgG2a control antibody (Miltenyi Biotec, Macquarie Park, NSW, Australia; cat# 130-106-546, RRID:AB_2661589); PE-conjugated mouse anti-STAT5 (pY694) antibody (BD Biosciences, Macquarie Park, NSW, Australia; cat# 612567, RRID:AB_399858); PE-conjugated rat anti-mouse CD123 (IL-3Rα) antibody (eBioscience, San Diego, CA, USA; cat# 12-1231-82, RRID:AB_465839); AF488 conjugated rat anti-mouse β IL-3 antibody (R&D Systems, Minneapolis, MN, USA; cat# FAB5492G, RRID: RRID:AB_2905558); Rat anti-mouse CD131 (mβc) antibody (BD Biosciences, cat# 740050, RRID:AB_2739817).

Techniques: Purification, Binding Assay, Expressing, Incubation, Fluorescence, Isolation, Produced