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: Annals of the rheumatic diseases

Article Title: Cathepsin S inhibition suppresses systemic lupus erythematosus and lupus nephritis because cathepsin S is essential for MHC class II-mediated CD4 T cell and B cell priming.

doi: 10.1136/annrheumdis-2013-203717

Figure Lengend Snippet: Figure 4 R05461111 reduces autoantibodies in MRL-Fas(lpr) mice. (A) Antinuclear antibodies (ANA) staining patterns on Hep2 human epithelial cells for serum derived from vehicle-treated and R05461111-treated MRL-Fas(lpr) mice at 1:200 dilution. (B) Crithidia luciliae slides were incubated with 1:50 diluted plasma of 20-week-old mice from both treatment groups, and autoantibody binding to the flagellate’s kinetoplast was detected using an FITC-labelled goat antimouse IgG. Images on the left show anti-dsDNA IgG in green, and staining the kinetoplast DNA itself with DAPI shown in blue is shown in the middle. The merged pictures are shown on the right side, demonstrating that FITC positivity matches with the kinetoplast at the flagella pole of C luciliae. (C) Plasma levels of anti-dsDNA IgG and IgM antibodies were determined by ELISA. (D) Plasma levels of IgG isotypes such as IgG1, IgG2a, IgG2b and IgG3 against ds-DNA were determined by ELISA. (E) Plasma levels of anti-RNP/Sm, antinucleosome antibodies and rheumatoid factor were determined by ELISA. Data are expressed as means±SEM (n=15 each treatment group,). *p<0.05, **p<0.01, ***p<0.001 versus vehicle.

Article Snippet: For the detection of IgG and its isotypes in the serum, plates were precoated with antimouse IgG, IgG1, IgG2a, IgG2b and IgG3 (Bethyl Laboratories, Montgomery, Texas, USA), and sera were applied at dilutions of 1:10 000 to 1:50 000.

Techniques: Staining, Derivative Assay, Incubation, Clinical Proteomics, Binding Assay, Enzyme-linked Immunosorbent Assay