Journal: Scientific reports

Article Title: A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies.

doi: 10.1038/s41598-021-84116-w

Figure Lengend Snippet: Figure 2. NIT211 conjugate is recognized by oligomannose-specific bnAbs and their germline precursors. NIT211 (4.1 glycosides per CRM197) was coated as solid-phase antigen onto ELISA plate wells at 5 µg/ml in PBS and assayed for recognition by the different antibodies. All antibodies were expressed recombinantly as human IgG1. (A) Binding of PGT128/130 bnAb family members PGT125, PGT126, PGT128 and PGT130. (B) Binding of non-PGT128/130 family antibodies BF520.1, BG18, PCDN-33A, PGDM12, PGDM21, PCDN76- 33A, VRC41.01. (C) Binding of inferred gl precursors BF520.1, BG18, PCDN-33A and the PGT128/130 family. Results are from single experiments performed with technical duplicates.

Article Snippet: Total serum IgG was detected with a mixture of equal amounts of alkaline phosphatase conjugated anti-mouse IgG1, IgG2b, IgG2c and IgG3 secondary antibodies (Jackson ImmunoResearch) and nitrophenylphosphate substrate (Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Scientific reports

Article Title: A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies.

doi: 10.1038/s41598-021-84116-w

Figure Lengend Snippet: Figure 4. The relative binding affinities of PGT antibodies for NIT211 increase with increasing ligand density. Binding to NIT211 conjugates was assessed by ELISA. NIT211 (2.6 ligands, 4.1, and 6.2 ligands) was coated as solid-phase antigen onto ELISA plate wells at 5 µg/ml (82, 79, 76 nM respectively) and assayed for antibody binding. (A) Binding of PGT125, PGT126, PGT128 and PGT130 IgG to NIT211 at three different densities of glycoside per CRM197. (B) Binding of PGT125, PGT126, PGT128 and PGT130 Fabs to NIT211 at two different densities of glycoside per CRM197.

Article Snippet: Total serum IgG was detected with a mixture of equal amounts of alkaline phosphatase conjugated anti-mouse IgG1, IgG2b, IgG2c and IgG3 secondary antibodies (Jackson ImmunoResearch) and nitrophenylphosphate substrate (Sigma).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies.

doi: 10.1038/s41598-021-84116-w

Figure Lengend Snippet: Figure 5. Only animals administered GLA-SE-adjuvanted NIT211 mount an IgG response to the oligomannose mimetic that is of the IgG3 subclass. Trianni mice (n = 5 per group) were immunized subcutaneously (days 0, 21, 42 and 105) and sera collected on day 0 prior to immunization and on days 10, 28, 49, and 119 post- immunization. (A) Binding of total IgG antibodies in pre-immune and post-immune sera to BSA-conjugated oligomannose mimetic. Binding curves represent mean values for the five animals in each immunization group, each tested in duplicate, with error bars denoting the standard deviation from the mean. (B) Individual IgG binding curves for NIT211 + GLA-SE immunized mice (Ms1 to 5). The sex of each mouse is also noted. (C) IgG1, IgG2b, IgG2c and IgG3 antibody subclass responses in day 119 post-immune sera of NIT211 + GLA-SE immunized animals for the BSA-conjugated oligomannose mimetic in comparison to the CRM197 protein carrier.

Article Snippet: Total serum IgG was detected with a mixture of equal amounts of alkaline phosphatase conjugated anti-mouse IgG1, IgG2b, IgG2c and IgG3 secondary antibodies (Jackson ImmunoResearch) and nitrophenylphosphate substrate (Sigma).

Techniques: Binding Assay, Standard Deviation, Comparison

Journal: Scientific reports

Article Title: A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies.

doi: 10.1038/s41598-021-84116-w

Figure Lengend Snippet: Figure 6. NIT211 formulated in GLA-SE elicits antibodies with capacity to bind recombinant HIV Env trimers. (A) Binding of total IgG from day 119 sera from unimmunized, and NIT211-immunized animal groups to SOSIP trimers AMC008 (subtype B), Du422 (subtype C), ZM197M (subtype C), BG505 (subtype A), B41 (subtype B), and CZA97 (subtype C). The HIS-tagged trimers were captured on nickel-coated ELISA plates at 5 µg/ml in PBS. (B) Level of IgG1, IgG2, IgG3 antibody subclass binding (1:100 dilution) to B41 SOSIP trimer in day 119 sera of NIT211 + GLA-SE immunized animals. (C) Residual binding of bnAb PGT128 to B41 SOSIP trimer following incubation with sera from NIT211 + GLA-SE immunized animals (day 119) vs sera from KLH + Alum/CpG ODN1826 immunized animals (day 34). (D) Day 119 sera from NIT211 + GLA-SE immunized animals and sera from a group of unimmunized animals were assessed for pseudovirus neutralization using a panel of seven diverse HIV-1 strains (92TH021, 92RW020, 94UG103, 92BR020, 97ZA012, JRCSF and NL4-3). Pseudotyped vesicular stomatitis virus (VSV) was used as a negative control. All graphs depict mean values for the serum samples from all animals (n = 5) in each group. Error bars represent standard error from the mean.

Article Snippet: Total serum IgG was detected with a mixture of equal amounts of alkaline phosphatase conjugated anti-mouse IgG1, IgG2b, IgG2c and IgG3 secondary antibodies (Jackson ImmunoResearch) and nitrophenylphosphate substrate (Sigma).

Techniques: Recombinant, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Neutralization, Virus, Negative Control

Journal: Differentiation; research in biological diversity

Article Title: The combination of glycosaminoglycans and fibrous proteins improves cell proliferation and early differentiation of bovine primary skeletal muscle cells.

doi: 10.1016/j.diff.2013.06.006

Figure Lengend Snippet: Fig. 2. Localization and expression of myogenic regulatory transcription factors (MRFs) during proliferation and differentiation. (A) Proliferating cells were fixed with 2% PFA, immunostained with Alexa 488-Phalloidin (green), and rabbit anti-MyoD, followed by DyLight 649-conjugated donkey anti-rabbit (red) before fluorescence microscopy analysis. Nuclei were stained with DAPI (blue). Scale bar 10 mM. (B) Cells differentiated for 3 days were fixed with 2% PFA, immunostained with Alexa 488-Phalloidin (green), and goat anti-Myogenin, followed by DyLight 649-conjugated donkey anti-goat (red) before fluorescence microscopy analysis. Nuclei were stained with DAPI (blue). Arrows indicate Myogenin positive cells. Scale bar 10 mM. (C) Differentiating cells were fixed with 2% PFA, immunostained with Alexa 488-Phalloidin (green) and rabbit anti-Desmin, followed by DyLight 549-conjugated mouse anti-rabbit (red) before fluorescence microscopy analysis. Nuclei were stained with DAPI (blue). Scale bar 10 mM. (D) Left: a representative western blot showing the expression level of MyoD and Desmin after 0 and 3 days of differentiation. Cell lysates were subjected to western blotting using antibodies to MyoD, Desmin, and Tubulin (loading control). Right: quantification of Western blots. Bars show the expression of Desmin and MyoD, relative to tubulin, after 0 and 3 days of differentiation. The graph represents the average of three experiments with SD.

Article Snippet: Alexa 488-conjugated goat anti-mouse and DyLight 549- conjugated mouse anti-rabbit were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

Techniques: Expressing, Microscopy, Staining, Western Blot, Control

Journal: Differentiation; research in biological diversity

Article Title: The combination of glycosaminoglycans and fibrous proteins improves cell proliferation and early differentiation of bovine primary skeletal muscle cells.

doi: 10.1016/j.diff.2013.06.006

Figure Lengend Snippet: Fig. 6. Desmin staining of myotubes on different surface coatings after 1, 3 or 5 days in differentiation medium. Differentiating cells were fixed with 2% PFA and immunostained with rabbit anti-Desmin, followed by DyLight 549-conjugated mouse anti-rabbit (yellow) before fluorescence microscopy analysis. Nuclei were stained with DAPI (blue). Scale bar 20 mM.

Article Snippet: Alexa 488-conjugated goat anti-mouse and DyLight 549- conjugated mouse anti-rabbit were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

Techniques: Staining, Microscopy