Journal: International immunology

Article Title: Regulation of NOD mouse autoimmune diabetes by T cells that recognize a TCR CDR3 peptide.

doi: 10.1093/intimm/11.6.957

Figure Lengend Snippet: Fig. 8. Vaccination to C9 CDR3β induces anti-C9 CDR3β IgG2a antibodies and anti-p277 IgG2b and IgG1 antibodies. Ten-week-old female NOD mice were vaccinated with flagellin (filled shapes) or with recombinant C9 CDR3β–flagellin (open shapes). Four weeks later, the sera of individual mice were assayed by ELISA for antibody isotypes to C9 CDR3β peptide (upper panel) or to p277 peptide (lower panel). The response to C9 CDR3β was significantly of the IgG2a isotype (P , 0.01), while the response to p277 was significantly of the IgGI (P , 0.05) and IgG2b isotypes (P , 0.01).

Article Snippet: The binding of antibodies to the adherent antigens was detected using alkaline phosphatase-conjugated antimouse IgG 1 IgM, or isotype-specific anti-mouse IgG1, IgG2a or IgG2b (Jackson Immunoresearch, West Grove, PA).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: Regulation of NOD mouse autoimmune diabetes by T cells that recognize a TCR CDR3 peptide.

doi: 10.1093/intimm/11.6.957

Figure Lengend Snippet: Fig. 9. Vaccination to C9 CDR3β down-regulates the spontaneous antibody responses to intact hsp60 (A) and to insulin (B). Ten-week- old female NOD mice were vaccinated to flagellin or to recombinant C9 CDR3β–flagellin. Four weeks later, their serum IgG antibodies were assayed by ELISA to intact hsp60 or to insulin. The antibody titers of the C9 CDR3β-vaccinated mice were significantly decreased (*P , 0.01).

Article Snippet: The binding of antibodies to the adherent antigens was detected using alkaline phosphatase-conjugated antimouse IgG 1 IgM, or isotype-specific anti-mouse IgG1, IgG2a or IgG2b (Jackson Immunoresearch, West Grove, PA).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: An expressed neo(r) cassette provides required functions of the 1gamma2b exon for class switching.

doi: 10.1093/intimm/10.11.1683

Figure Lengend Snippet: Fig. 1. IgG2b I exon and splice donor targeting of F1 and homozygous ES cells. (A) Construct. Approximately 290 bp of the I exon (SacI–HincII) along with 77 bp downstream of the splice donor site are deleted. Triangles represent loxP sites. The first 110 bp and the Iγ2b promoter are left intact to retain most of the transcription initiation sites (five out of seven retained). Restriction site abbreviations: S, SacI; H, HindIII; B, BamHI; H2, HincII. pIγ2b refers to the endogenous promoter for Iγ2b. (B) The targeting construct 6UTlN1X was transfected into ES cells and targeted cells were identified by Southern blot analysis using probe 1. The Cre recombinase gene was transiently transfected into ES cells homozygous for the PGK-neor gene to delete the neor gene.

Article Snippet: ELISA analysis Splenocyte culture supernatants from day 4.5 or 5 and sera were analyzed for the presence of different Ig isotypes by sandwich ELISA assay as previously described (13,28) using goat anti-mouse IgG2b coating (Southern Biotechnology Associates) and alkaline phosphatase-labeled anti-mouse IgG2b (R12-3) (PharMingen).

Techniques: Construct, Transfection, Southern Blot

Journal: International immunology

Article Title: An expressed neo(r) cassette provides required functions of the 1gamma2b exon for class switching.

doi: 10.1093/intimm/10.11.1683

Figure Lengend Snippet: Fig. 2. In vitro analysis of splenocytes from Iγ2b-targeted chimeric mice. (A) FACS analysis of IgG2b surface expression in mutant and control mice on day 4.5 of splenocytes cultured in RPMI/10% FCS medium containing LPS (20 µg/ml) with or without rIL-4 (20 ng/ml). One of five experiments is shown. Percents are calculated out of total live lymphocytes. (B) ELISA analysis of culture supernatants from splenic B cells stimulated in vitro with LPS or LPS 1 IL-4 (summary of three experiments from Fig. 2A). Open squares are LPS, filled squares are LPS 1 IL-4. (C) ELISA analysis of IgG2b antibody serum levels of mutant and control mice.

Article Snippet: ELISA analysis Splenocyte culture supernatants from day 4.5 or 5 and sera were analyzed for the presence of different Ig isotypes by sandwich ELISA assay as previously described (13,28) using goat anti-mouse IgG2b coating (Southern Biotechnology Associates) and alkaline phosphatase-labeled anti-mouse IgG2b (R12-3) (PharMingen).

Techniques: In Vitro, Expressing, Mutagenesis, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

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

Article Title: DAXX represents a new type of protein-folding enabler.

doi: 10.1038/s41586-021-03824-5

Figure Lengend Snippet: Fig. 1 | DAXX prevents protein misfolding and aggregation. a, b, Heat-induced luciferase inactivation (a, 5 nM) and aggregation (b, 200 nM) in presence or absence of GST, DAXX and HSP70–HSP40 at 200 nM (a) or at the indicated molar ratios (b). c, Aggregation of ATXN1(82Q) (50 nM) in the presence or absence of glutathione S-transferase (GST) or DAXX (200 nM each). Raw data for this and other gels are found in Supplementary Fig 1. PE, pelletable aggregates that were soluble with SDS; SN, supernatant. d–f, Fibrillization of α-Syn (70 μM) in the presence GST, HSP70–HSP40, HSPs (HSP70–HSP40–HSP104(A503S)) (200 nM each) and DAXX (100–400 nM) was assayed by ThT-binding (d), electron microscopy (e; red arrows, fibrils; blue arrows, large oligomers; scale bar, 100 nm), and sedimentation followed by dot blot for SDS-soluble and SDS-resistant (SR) aggregates and total α-Syn and by disuccinimidyl suberate (DSS) cross-linking for soluble oligomers (f). IB, immunoblot. g–i, Fibrillization of Aβ42 monomers (10 μM) in the absence or presence of DAXX (50–600 nM) (g, h), and viability of SH-SY5Y cells treated with Aβ42 pre-incubated with or without DAXX (i). An ATP-regeneration system was included with heat-shock proteins but not DAXX (all subsequent experiments with heat-shock proteins, but not experiments with DAXX, also contained an ATP-regeneration system). Data are mean or mean ± s.d. (n = 4 for i, and 3 for the rest) and are representative of three independent experiments. *P < 0.05, NS, not significant; unpaired Student’s t-test.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Antibodies against the following proteins/epitopes were purchased from the indicated sources: GAPDH (sc-47724,Mouse, WB, IP, IF and IHC(P)), His (sc-8036, Mouse, IP, WB, IHC(P), ELISA, IF, FCM), GST (sc-138, Mouse, IP, WB, IHC(P), ELISA, IF, FCM), p53DO1 (sc-126, Mouse, WB, IP, IF, IHC(P) and FCM), Mdm2 (sc-965, Mouse, WB, IP, IF and IHC(P)), DAXX (sc-8043, Mouse IP,WB, IHC(P), ELISA, IF, FCM), α-synuclein syn211 (sc-12767, Mouse, IP, WB, IHC(P), ELISA, IF, and FCM) (Santa Cruz Biotechnology); Flag (#14793, Rabbit, WB, IP, IHC, ChIP, IF, FCM, ELISA) and DAXX (#4533S, Rabbit, WB, IP, IHC, ChIP, IF, FCM, ELISA) (Cell Signaling Technology); p53 (PAb1620, #OP33, Mouse, WB, IP, IF; PAb240, #OP29, Mouse, WB, IP), and Mdm2 (#OP46, Mouse, WB, IP) (Calbiochem); and HA (ab137838, Rabbit, WB, ICC/IF, IP) and Luciferase (ab21176, Rabbit, ICC/IF, WB) (Abcam); βAmyloid 1-42 (#805509) (BioLegend, Mouse, WB, IHC(P), ELISA).

Techniques: Luciferase, Binding Assay, Electron Microscopy, Sedimentation, Dot Blot, Western Blot, Incubation

Journal: iScience

Article Title: Targeting B cells in the pre-phase of systemic autoimmunity globally interferes with autoimmune pathology.

doi: 10.1016/j.isci.2021.103076

Figure Lengend Snippet: Figure 4. Effect of pre-phase B cell depletion on humoral immunity (A) Shown is the total amount of serum IgM, IgG1, IgG2b, IgG2c, IgG3 and IgA in 10- and 18-week-old anti-CD20 (aCD20) or isotype control antibody treated female (\) and male (_) BXSB mice as determined by quantitative ELISA. Significant differences between indicated groups were tested with ordinary one-way ANOVA with Tukey’s multiple comparison test (normal distribution) or Kruskal-Wallis plus Dunn’s multiple comparison test (no normal distribution). Two independent experiments with a total of n = 5–12 mice per group. Symbols represent individual mice; error bars represent the mean G SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: For quantification of total serum IgM, IgG and IgA the Mouse IgM, IgG and IgA ELISA Quantification Kits (Bethyl) were used according to the manufacturer’s instructions: ELISA plates were coated with 100 ng/well goat anti-mouse IgM, IgG or IgA in Carbonate/Bicarbonate for 1 h at room temperature.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Comparison