Journal: PLoS ONE

Article Title: Immunogenicity of Isogenic IgG in Aggregates and Immune Complexes

doi: 10.1371/journal.pone.0170556

Figure Lengend Snippet: (A) Adoptive transfer and immunization scheme. (B) Serum IgG anti-idiotypic antibodies directed against the Vκ of mAb 36–71 were quantified using DELFIA as described in Materials and Methods. Connected lines denote a single mouse. Data are representative of two independent experiments with n = 5 mice per group. (C) Table summarizing results from 2 experiments with n = 5 mice per group with positive titers at each time point by treatment. Both experiments had 3 mice with positive titers at d42 and 5 mice with positive titers at d63 in both the heat-aggregated and immune complex groups.

Article Snippet: For the anti-CD20 B cell depletion experiment, mice were injected i.p. with 500 μg of 5D2, an anti-mouse CD20 IgG2a/κ or control anti-ragweed monoclonal IgG2a/κ both generously donated by Genentech (San Francisco, CA) in sterile low aggregation pharmaceutical buffer 24 hours prior to i.v. adoptive transfer (day -2) of 5 x 10 4 CFSE-labeled T cells and 48 h prior to antigen injection.

Techniques: Adoptive Transfer Assay

Journal: PLoS ONE

Article Title: Immunogenicity of Isogenic IgG in Aggregates and Immune Complexes

doi: 10.1371/journal.pone.0170556

Figure Lengend Snippet: (A) Adoptive transfer and immunization scheme. Primary and secondary injections were as specified in the figure. Naïve mice were B6AF1 mice that received CA30 cells, but no primary or secondary injection. (B) Mean titration curves (above) and concentrations (below) of serum IgG anti-Id at day 21. Each point represents an experimental mouse (n = 5/group). Results representative of 2 independent experiments with n = 5 mice per group.

Article Snippet: For the anti-CD20 B cell depletion experiment, mice were injected i.p. with 500 μg of 5D2, an anti-mouse CD20 IgG2a/κ or control anti-ragweed monoclonal IgG2a/κ both generously donated by Genentech (San Francisco, CA) in sterile low aggregation pharmaceutical buffer 24 hours prior to i.v. adoptive transfer (day -2) of 5 x 10 4 CFSE-labeled T cells and 48 h prior to antigen injection.

Techniques: Adoptive Transfer Assay, Injection, Titration

Journal: The Journal of biological chemistry

Article Title: Rapid, scalable assay of amylin-β amyloid co-aggregation in brain tissue and blood.

doi: 10.1016/j.jbc.2023.104682

Figure Lengend Snippet: Figure 1. Detection of amylin-Aβ hetero-oligomers in human AD brain tissues. A, the schematic illustration of amylin-Aβ hetero-amyloid formation in the brain along with an example of cerebral amylin-Aβ codeposition reported previously (12). The section through the brain of a PS1 mutation carrier shows cerebral Aβ deposits (green) and amylin (brown) forming the plaque core and intercalated amylin-Aβ deposits. B, confocal microscopic analysis of a section through the brain of PS1 mutation carrier stained with Thioflavin S (Thio S; green), amylin (red), and Aβ (magenta). C, confocal microscopic analysis of a section through the brain of a person with sAD stained with amylin (green) and Aβ (red) and further subjected to an amylin-Aβ PLA (red). D, schematic representation of an amylin-Aβ sandwich ELISA with the anti-amylin antibody used as the capture antibody and anti-Aβ antibody conjugated with horseradish peroxidase (HRP) used as the detection antibody. E, the amino acid sequence analysis of amylin and Aβ peptides indicating the epitope regions against the anti-amylin capture and anti-Aβ detection antibodies used in the amylin-Aβ sandwich ELISA. F, amylin-Aβ immunoreactivity signal intensity measured in human sAD brain tissue homogenates (n = 5) using the P2 amylin antibody as the capture antibody, and total Aβ or Aβ42 or Aβ40 antibodies as the detection antibody in the amylin-Aβ sandwich ELISA. Scale bars represent 10 μm in (B) and 20 μm in (C). Aβ, β amyloid; AD, Alzheimer’s disease; PLA, proximity ligation assay; PS1, presenilin 1; sAD, sporadic Alzheimer’s disease.

Article Snippet: Briefly, rat pancreas homogenates (1000 μg of total protein) and amylinAβ (30 μM amylin and 16.2 μM Aβ; 1:1) aggregates were incubated with rabbit anti-amylin P2 antibody (1:25, 2 mg/ml stock) and normal rabbit Ig G (#2729; Cell Signaling Technology) as a control, overnight with end-over-end rotation, at 4 C. Antigen–antibody complex was added to Immobilized Protein A/G resin slurry (20422, Thermo Fisher Scientific) for 2 h at ambient temperature, washed with wash buffer (5 mM of EGTA, 50 mM of Tris, 1% v/v of Triton X-100, pH 7.5 + 1% v/ v protease inhibitor, 1% v/v phosphatase inhibitor) and samples eluted with elution buffer (1.5% w/v of glycine, 8% v/v of 1 N HCl, pH 2–3) from the resins using elution buffer.

Techniques: Mutagenesis, Staining, Sandwich ELISA, Sequencing, Proximity Ligation Assay

Journal: The Journal of biological chemistry

Article Title: Rapid, scalable assay of amylin-β amyloid co-aggregation in brain tissue and blood.

doi: 10.1016/j.jbc.2023.104682

Figure Lengend Snippet: Figure 2. Specificity and sensitivity of the amylin-Aβ sandwich ELISA to detection of amylin-Aβ hetero-oligomers. A, the average amylin-Aβ immunoreactivity signal intensities measured in amylin-Aβ (30 μM amylin and 16.2 μM Aβ; mixed in a 1:1 ratio to give final concentrations of 15 μM amylin and 8.1 μM Aβ), amylin (30 μM), and Aβ (16.2 μM) aggregates using the amylin-Aβ sandwich ELISA. B, thioflavin T (Th-T) fluorescence signal intensities measured in the solutions of human amylin-Aβ and rat amylin-Aβ aggregates (same peptide concentrations as in A). C, amylin-Aβ immunoreactivity signal intensities measured by the amylin-Aβ sandwich ELISA in rat amylin-Aβ and human amylin-Aβ hetero-oligomers. D, amylin-Aβ immunoreactivity signal intensities measured by the amylin-Aβ sandwich ELISA in amylin-Aβ dilutions (1:10–1:160) using the same aliquots of amylin-Aβ as in (B). E, coimmuno- precipitation of amylin using the P2 amylin antibody in amylin-Aβ aggregates (undiluted and 1:64 diluted) followed by Western blot analysis of Aβ and amylin in amylin IP eluates. F, amylin-Aβ immunoreactivity signal intensities measured by the amylin-Aβ sandwich ELISA in the fraction collected at various time points of human amylin (30 μM) incubated with human Aβ40 (16.2 μM). G, Th-T fluorescence signal intensities measured in the same fraction collected as in (F). H, pairwise correlation analyses of amylin-Aβ signal intensity measured in (F) versus Th-T fluorescence signal intensities measured in (G). Data are mean ± SD. Data are presented as correlation analysis, Pearson’s correlation **p < 0.01 in (H). Aβ, β amyloid.

Article Snippet: Briefly, rat pancreas homogenates (1000 μg of total protein) and amylinAβ (30 μM amylin and 16.2 μM Aβ; 1:1) aggregates were incubated with rabbit anti-amylin P2 antibody (1:25, 2 mg/ml stock) and normal rabbit Ig G (#2729; Cell Signaling Technology) as a control, overnight with end-over-end rotation, at 4 C. Antigen–antibody complex was added to Immobilized Protein A/G resin slurry (20422, Thermo Fisher Scientific) for 2 h at ambient temperature, washed with wash buffer (5 mM of EGTA, 50 mM of Tris, 1% v/v of Triton X-100, pH 7.5 + 1% v/ v protease inhibitor, 1% v/v phosphatase inhibitor) and samples eluted with elution buffer (1.5% w/v of glycine, 8% v/v of 1 N HCl, pH 2–3) from the resins using elution buffer.

Techniques: Sandwich ELISA, Western Blot, Incubation