Journal: ACS infectious diseases

Article Title: Tyrosine-Based Cross-Linking of Peptide Antigens to Generate Nanoclusters with Enhanced Immunogenicity: Demonstration Using the Conserved M2e Peptide of Influenza A.

doi: 10.1021/acsinfecdis.1c00219

Figure Lengend Snippet: Figure 6. In vitro antigenicity analysis of t-M2e-t NCs using ELISA. (A) Absorbance values from an indirect ELISA performed by coating 96-well plates with a fixed amount of either un-cross-linked t-M2e-t peptide, cross-linked t-M2e-t NCs, or a negative control peptide (NCP). Mouse serum collected from mice vaccinated with a gold nanoparticle and M2e formulation (from another study8−10) was used as a source of M2e detection IgG antibody, which was applied at different dilutions. (B) Absorbance values from a sandwich ELISA performed by coating 96-well plates with a fixed amount of purified mouse anti-M2e IgM monoclonal antibody as capture antibody. Dilutions of analyte (either un-cross-linked t-M2e-t peptide, cross-linked t-M2e-t NCs, or a NCP) were then added to the wells. Mouse serum collected from mice vaccinated with a gold nanoparticle and M2e formulation (from another study8−10) was used as a source of primary M2e detection IgG antibody. Cartoons of each ELISA procedure are shown below their respective graphs.

Article Snippet: All peptides were end-terminal acetylated and amidated in order to increase peptide stability against degradation.65−67 Goat anti-mouse IgG (1030−05), goat anti-mouse IgG1 (1070−05), and goat anti-mouse IgG2a (1080−05) secondary antibodies with HRP were obtained from Southern Biotech (AL, USA).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Indirect ELISA, Negative Control, Formulation, Sandwich ELISA

Journal: ACS infectious diseases

Article Title: Tyrosine-Based Cross-Linking of Peptide Antigens to Generate Nanoclusters with Enhanced Immunogenicity: Demonstration Using the Conserved M2e Peptide of Influenza A.

doi: 10.1021/acsinfecdis.1c00219

Figure Lengend Snippet: Figure 7. Immune response and survival data of mice after immunization with t-M2e-t NCs. Mice were immunized thrice, once each on day 0, 21, and 42. Blood was collected and analyzed for anti-M2e antibodies. Within 1 week of the final serum collection, mice were challenged with 3× LD50 A/California/07/2009 (H1N1) and monitored for 14 days. Two antigens were used (i) 20 μg or 5 μg t-M2e-t UCP (un-cross-linked peptide) with/without 20 μg CpG, and (ii) 20 μg or 5 μg S t-M2e-t NCs (small NCs) with/without 20 μg CpG. (A−C) Anti-M2e IgG, IgG1 and IgG2a titers with t-M2e-t UCP as antigen. (D−F) Anti-M2e IgG, IgG1 and IgG2a titers with t-M2e-t NC as antigen. (G, H) Body weight and survival after virus challenge of t-M2e-t UCP groups. (I, J) Body weight and survival after virus challenge of t-M2e-t NP groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: All peptides were end-terminal acetylated and amidated in order to increase peptide stability against degradation.65−67 Goat anti-mouse IgG (1030−05), goat anti-mouse IgG1 (1070−05), and goat anti-mouse IgG2a (1080−05) secondary antibodies with HRP were obtained from Southern Biotech (AL, USA).

Techniques: Virus

Journal: Nature communications

Article Title: A child with perinatal HIV infection and long-term sustained virological control following antiretroviral treatment cessation.

doi: 10.1038/s41467-019-08311-0

Figure Lengend Snippet: Fig. 3 HIV-specific responses and immune response capability of the case at 9.5 years of age. a Detection of HIV-specific antibodies at 9.5 years of age by western blot. The case antibody profile is compared with controls that are a high positive, low positive and HIV-negative. HIV proteins corresponding to bands in the blots are shown in the left grey-shaded block; the case profile was positive for the core proteins indicated in pink. b Quantitation of HIV- specific antibodies by multiplex bead array for all isotypes and subclasses (indicated on the left side—IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2) against gp41, Gag, RT, gp120, Nef, Tat, Vpu, peptide Vpu9 and V1V2 scaffold antigens (indicated at the top). Results are expressed as mean fluorescence intensities (MFI)), the colour key shows ranges of MFI according to colour intensity (the darker the more HIV-specific antibody detected). A result is considered positive if above the cut-off (mean ± 3 SD) determined from eight adult uninfected controls. Vpu9 amino acid sequence: STMVDMGHLRLLDVNDL. c Proportions of natural killer (NK) cells that respond to anti-CD16 antibody, and CD4+ and CD8+ T cells that respond to staphylococcal enterotoxin B (SEB) in a whole blood intracellular cytokine (ICC) assay that measures induction of interferon-γ (IFN-γ) and interleukin-2 (IL-2). HIV-uninfected adult reference values for comparison (n = 21; median % and range)—natural killer (NK) anti-CD16%: 37.92 (12–67.6), CD4 SEB%: 6.04 (0.25–11.91), CD8 SEB%: 5.82 (0.18–18.94). d A weak positive CD4+ T cell response to Gag (0.116%) in the absence of a detectable CD8+ T cell response to Gag (<0.1%; 0.023%). UN: addition of costimulatory antibodies anti-CD28 and anti-CD49d, no stimulation with peptides

Article Snippet: HIV-specific antibody isotypes were detected by adding 50 μL per well at 2 μgmL−1, R-phycoerthyrinconjugated mouse anti-human IgG1 to IgG4 (Cat. Nos 9052-09, 9070-09, 9210-09, 9200-09, respectively, Southern Biotech, USA), mouse anti-human IgM (Cat. No. 9020-09, Southern Biotech), mouse anti-human IgA1 (Cat. No. 9130-09, Southern Biotech) or mouse anti-human IgA2 (Cat. No. 9140-09, Southern Biotech) with shaking (in the dark at room temperature) followed by three washes.

Techniques: Western Blot, Blocking Assay, Quantitation Assay, Multiplex Assay, Sequencing, Immunocytochemistry, Comparison

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

doi: 10.1038/labinvest.2010.168

Figure Lengend Snippet: Figure 1 Semiquantitative RT-PCR and real-time PCR for Furin, TACE and AREG genes expression. (a) RT-PCR analysis of Furin, TACE and AREG mRNA extracted from SGEC treated with anti-Ro/SSA autoantibodies. M, marker; control, untreated SGEC; HIgG, SGEC treated with IgG fractions extracted from sera of healthy donors; anti-Ro, SGEC treated with anti-Ro/SSA autoantibodies. RT-PCR of GADPH was used as control. Band intensities were analyzed by densitometry (b). (c) Real-time PCR for Furin, TACE and AREG genes expression. Representative histograms of mRNA levels of Furin, TACE and AREG in untreated SGEC (control), SGEC treated with healthy IgG (HIgG), anti-Ro/SSA autoantibodies (anti-Ro). The mRNA levels of the housekeeping gene, b-2 microglobulin, were quantified between untreated control cells and variously treated cells (data represent the mean±s.e. of five independent experiments).

Article Snippet: Membranes were incubated for 90 min with rabbit anti-human Furin polyclonal antibody (pAb), goat antihuman TACE pAb (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human AREG mAb (R&D Systems, Minneapolis, MN USA), and for 30 min with the relative secondary antibodies-HRP conjugates (Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Marker, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

doi: 10.1038/labinvest.2010.168

Figure Lengend Snippet: Figure 2 Analysis of Furin, TACE and AREG expression in anti-Ro/SSA Abs-treated SGEC. (a) Flow cytometric analysis of Furin, TACE and AREG expression in SGEC after anti-Ro/SSA Abs treatment. Examples of flow cytometric images from one representative experiment. (A) Furin expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC; (B) intracellular active TACE expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC; (C) AREG expression analysis in untreated and anti-Ro/SSA or HIgG-treated SGEC. (b) Western blot analysis of Furin, TACE and AREG proteins expression in SGEC treated or not with anti-Ro/SSA. Immunoblotting gave rise to bands of the expected size (97 kDa for Furin, 80 kDa for active TACE and 50 kDa for AREG). b-Actin was used as protein loading control. (c) Detection of soluble AREG by ELISA. Secreted AREG was detected by ELISA in the conditioned medium. Control, untreated SGEC; HIgG, SGEC treated with IgG fractions extracted from sera of healthy donors; anti-Ro, SGEC treated with anti-Ro/SSA autoantibodies. (Data represent the mean±s.e. of four independent experiments).

Article Snippet: Membranes were incubated for 90 min with rabbit anti-human Furin polyclonal antibody (pAb), goat antihuman TACE pAb (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human AREG mAb (R&D Systems, Minneapolis, MN USA), and for 30 min with the relative secondary antibodies-HRP conjugates (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay