viral hpv protein  (Bio-Techne corporation)

Journal: medRxiv

Article Title: Development of HPV 16/18 E6 oncoprotein paper-based nanokit for enhanced detection of HPV 16/18 E6 oncoprotein in cervical cancer screening

doi: 10.1101/2020.04.29.20084459

Figure Lengend Snippet:

Article Snippet: The recombinant viral HPV protein (Lot # 25158317A, Cat. # AP-120, 0.5mg/ml bio-techne an R&D Systems Company) was diluted into the concentrations indicated in .

Techniques: Recombinant, Purification

Journal: Frontiers in Immunology

Article Title: Engineered human B cells targeting tumor-associated antigens exhibit antigen presentation and antibody-mediated functions

doi: 10.3389/fimmu.2025.1621222

Figure Lengend Snippet: Engineered B cells activate BCR signaling cascades and secrete isotype-switched antigen-specific antibodies. (A) Representative immunoblot of B cells engineered to express either C1P5 or 6F4 E6-binding BCRs, stimulated with recombinant E6 protein, anti-IgM/IgG protein control or unstimulated. Protein extracts were analyzed using anti-pERK antibody (top panel) or anti-total ERK antibody (bottom panel). (B) Quantification of mean ratio expression of ERK to pERK as in (A) ns=pv>0.05, *=pv<0.05, **=pv<0.01 for two-way ANOVA with Dunnett’s multiple comparisons test. (C) Same as A for the CLDN6-binding BCRs AB37 or IMAB206. Cell stimulation occurred with recombinant CLDN6 protein. (D) Quantification of experiments as in (C) For (A-D) , data representative of 2–3 experiments with 2 donor cells. ns=pv>0.05, **=pv<0.01 for two-way ANOVA with Dunnett’s multiple comparisons test. (E) Quantification of IgG antibodies in the supernatants of engineered B cells expressing either C1P5, AB37 or IMAB206, by ELISA. ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. (F) Representative ELISPOT images of E6-specific IgM, IgG and IgA secreted from B cells engineered to express the anti-E6 6F4 antibody. (G) Quantification of data as in (F) as normalized to the total amount of cells seeded in the wells. ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. For (E-G) pooled data from 2–3 experiments with 2–4 donor cells. For (B, D, E) , (G) mean and error bars are indicated, each dot represents an independent experiment.

Article Snippet: Recombinant E6 protein was from R&D Systems.

Techniques: Western Blot, Binding Assay, Recombinant, Control, Expressing, Cell Stimulation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Frontiers in Immunology

Article Title: Engineered human B cells targeting tumor-associated antigens exhibit antigen presentation and antibody-mediated functions

doi: 10.3389/fimmu.2025.1621222

Figure Lengend Snippet: Engineered B cells and immune complexes taken up by dendritic cells present E6 antigen to activate E6-specific MHC class II-restricted T cells. (A) Schema depicting the engineered B cell antigen presentation assay. B cells, either non-engineered (EP-only) or engineered to express anti-E6 antibodies (6F4 or C1P5), are loaded with recombinant E6 antigen at multiple concentrations and then incubated with T cells, either engineered (Class II TCR) or non-engineered (UTD) to express an anti-E6 TCR. The concentration of IFNγ in the supernatants is then analyzed by ELISA. (B) ELISA for IFNγ as in (A) Data representative of 2 experiments with 2 donors. Bars and error bars represent mean and SD. ns=pv>0.05, ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. (C) Schema depicting the immune complex-mediated antigen presentation assay. Immune complexes were formed with concentrated antibodies from the supernatants of B cells, either non-engineered or engineered to express anti-E6 antibodies, and recombinant E6 antigen at multiple concentrations. These were then incubated with myeloid-derived dendritic cells and then co-cultured with T cells, either engineered or non-engineered to express an anti-E6 TCR. The concentration of IFNγ in the supernatants is then analyzed by ELISA. (D) ELISA for IFNγ as in (C) Data representative of 2 experiments with 2 donors. Bars and error bars represent mean and SD. ns=pv>0.05, *=pv<0.05, ***=pv<0.001, ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. (E) Schema depicting the flow cytometry-based engineered B cell membrane-bound antigen uptake assay. Engineered or non-engineered B cells are incubated with labeled tumor cells either expressing or not expressing the targeted antigen. The frequency of B cells with the tumor label is then analyzed by flow cytometry. (F) Flow cytometry results as in (E) for B cells engineered to express anti-CLDN6 IMAB206 (blue) or anti-E6 (6F4) antibodies (grey). ns=pv>0.05, ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. (G) Flow cytometry results as in (E) for B cells engineered to express anti-E6 6F4 antibodies (orange), or anti-CLDN6 IMAB206 antibodies (grey). ns=pv>0.05, **=pv<0.01, for two-way ANOVA with Tukey’s multiple comparisons test. (H) Schema depicting a flow cytometry-based engineered B cell antigen presentation assay. B cells, either non-engineered or engineered to express anti-E6 antibodies, were incubated with tumor cells, engineered to express the membrane-bound E6 antigen, and with T cells, either engineered or non-engineered to express an anti-E6 TCR. The frequency of T cells expressing intracellular IFNγ was then analyzed by flow cytometry. (I) Flow cytometry results as in (H) for B cells engineered to express anti-E6 6F4 (orange) antibodies. Data representative of 3 experiments using one donor. Bars and error bars represent mean and SD. ns=pv>0.05, ****=pv<0.0001, for two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Recombinant E6 protein was from R&D Systems.

Techniques: Immunopeptidomics, Recombinant, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Flow Cytometry, Membrane, Labeling, Expressing

Journal: Frontiers in Immunology

Article Title: Engineered human B cells targeting tumor-associated antigens exhibit antigen presentation and antibody-mediated functions

doi: 10.3389/fimmu.2025.1621222

Figure Lengend Snippet: Engineered B cells activate BCR signaling cascades and secrete isotype-switched antigen-specific antibodies. (A) Representative immunoblot of B cells engineered to express either C1P5 or 6F4 E6-binding BCRs, stimulated with recombinant E6 protein, anti-IgM/IgG protein control or unstimulated. Protein extracts were analyzed using anti-pERK antibody (top panel) or anti-total ERK antibody (bottom panel). (B) Quantification of mean ratio expression of ERK to pERK as in (A) ns=pv>0.05, *=pv<0.05, **=pv<0.01 for two-way ANOVA with Dunnett’s multiple comparisons test. (C) Same as A for the CLDN6-binding BCRs AB37 or IMAB206. Cell stimulation occurred with recombinant CLDN6 protein. (D) Quantification of experiments as in (C) For (A-D) , data representative of 2–3 experiments with 2 donor cells. ns=pv>0.05, **=pv<0.01 for two-way ANOVA with Dunnett’s multiple comparisons test. (E) Quantification of IgG antibodies in the supernatants of engineered B cells expressing either C1P5, AB37 or IMAB206, by ELISA. ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. (F) Representative ELISPOT images of E6-specific IgM, IgG and IgA secreted from B cells engineered to express the anti-E6 6F4 antibody. (G) Quantification of data as in (F) as normalized to the total amount of cells seeded in the wells. ****=pv<0.0001 for two-way ANOVA with Šídák’s multiple comparisons test. For (E-G) pooled data from 2–3 experiments with 2–4 donor cells. For (B, D, E) , (G) mean and error bars are indicated, each dot represents an independent experiment.

Article Snippet: For soluble controls, 1e5 B cells were added to a 96-well plate and incubated with 0 or 100 nM E6 protein (R&D Systems) or 1 μg/well E6 1–15 peptide (Genscript) for 20 minutes, followed by the addition of 1e5 UTD or E6 TCR T cells for a 1:1 ratio of B Cells:T cells.

Techniques: Western Blot, Binding Assay, Recombinant, Control, Expressing, Cell Stimulation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: medRxiv

Article Title: Development of HPV 16/18 E6 oncoprotein paper-based nanokit for enhanced detection of HPV 16/18 E6 oncoprotein in cervical cancer screening

doi: 10.1101/2020.04.29.20084459

Figure Lengend Snippet:

Article Snippet: To ensure optimization, small spots of 2 μl of the different concentrations of the recombinant viral HPV E6 protein sample were spotted onto the NC and MN paper strips at the center of the grid slowly ensuring about 2–4 mm diameter using narrow-mouth FinTip pipette tips (Thomas Scientific).

Techniques: Recombinant, Purification

Journal: medRxiv

Article Title: Development of HPV 16/18 E6 oncoprotein paper-based nanokit for enhanced detection of HPV 16/18 E6 oncoprotein in cervical cancer screening

doi: 10.1101/2020.04.29.20084459

Figure Lengend Snippet: (1) Malignant transformation of cells due to HPV infection, (2) Dysplasia becomes apparent at the cervical tissue during early stage (3) During this early stage Early oncogenic protein, E6 is released (4) the HPV 16/18 E6-HRP (CP 15)-AuNPS bioconjugate that was developed in the study is reacted with E6 oncogenic protein yielding (5) an immune complex (6) Addition of TMB or deposit on TMB immobilized template yields the colorimetric results

Article Snippet: To ensure optimization, small spots of 2 μl of the different concentrations of the recombinant viral HPV E6 protein sample were spotted onto the NC and MN paper strips at the center of the grid slowly ensuring about 2–4 mm diameter using narrow-mouth FinTip pipette tips (Thomas Scientific).

Techniques: Transformation Assay, Infection