Journal: Journal of Alzheimer's Disease

Article Title: One of the binding proteins for administered amyloid-β appears to be anti-Aβ IgG antibody in amyloid plaques

doi: 10.1177/13872877251389631

Figure Lengend Snippet: Specific binding of applied Bio42 to APs demonstrated by a tissue competition assay (A-I) and schematic summary of the results (J, K). (A) Labeling of APs (arrowheads) with Bio42 in a brain tissue from a dementia and CAA patient is presented. Binding of Bio42 to APs and artery walls (asterisks) was specific to Aβ 42 , as coincubation with non-biotinylated Aβ 42 peptide (Pep42) progressively attenuated the binding in dose-dependent manner (Pep42/Bio42 ratio: 0.01 for (B); 0.1 for (C); 1 for (D); and 10 for (E)). (F) No labeling was observed without Bio42 as a negative control . In contrast, coincubation with non-biotinylated Aβ 40 peptide (Pep40) had little effect on Bio42 binding (Pep40/Bio42 ratio: 0.1 for (G); 1 for (H); and 10 for (I)), unlike Pep42 (scale bar, 100 μm). A fixed amount of Bio42 (1 μg/ml) was coincubated stepwise with Pep42 (J) or Pep40 (K) in various ratios. The corresponding AP labeling results shown in (A-E) with Pep42 and in (G-I) with Pep40 are summarized in tables. AP labeling with Bio42 was dense in the absence of Pep42 but gradually became fainter as the Pep42/Bio42 ratio increased. Given that Bio42 and Pep42 specifically bind to the BP and that Pep40 is noncompetitive, dense AP labeling is expected to be invariably preserved, regardless of the Pep40/Bio42 ratio.

Article Snippet: The following well-characterized antibodies were used at the appropriate concentrations for immunohistochemical, immunofluorescence staining, or WB analyses: biotinylated anti-human IgG antibody (Vector Laboratories), anti-biotin antibody (Jackson ImmunoResearch), goat anti-human IgG antibody (Merck).

Techniques: Binding Assay, Competitive Binding Assay, Labeling, Negative Control

Journal: Journal of Alzheimer's Disease

Article Title: One of the binding proteins for administered amyloid-β appears to be anti-Aβ IgG antibody in amyloid plaques

doi: 10.1177/13872877251389631

Figure Lengend Snippet: Labeling of amyloid plaques (APs) by biotinylated synthetic Aβ 42 peptide (Bio42) visualized by diaminobenzidine (DAB). (A-D) Brain sections including APs and cerebral amyloid angiopathy (CAA) were incubated with 0.1 μg/ml, 1 μg/ml, 10 μg/ml, and 100 μg/ml of Bio42 and stained by DAB. Three representative arteries are marked as landmarks (asterisks) that appear in all serial sections. (A) APs and artery walls were labeled clearly with 0.1 μg/ml of applied Bio42. (B) With 1 μg/ml of applied Bio42 the same APs and artery walls were labeled more densely with higher background. In addition, a larger number of APs were observed (arrowheads). (C) In contrast, with 10 μg/ml of applied Bio42 the labelling density was markedly decreased. (D) Moreover, the labeling was absent with 100 μg/ml of applied Bio42. There is an optimal concentration of binding for applied Bio42 to APs (scale bar, 100 μm).

Article Snippet: The following well-characterized antibodies were used at the appropriate concentrations for immunohistochemical, immunofluorescence staining, or WB analyses: biotinylated anti-human IgG antibody (Vector Laboratories), anti-biotin antibody (Jackson ImmunoResearch), goat anti-human IgG antibody (Merck).

Techniques: Labeling, Incubation, Staining, Concentration Assay, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Antibody inhibition of properdin prevents complement-mediated intravascular and extravascular hemolysis 1

doi: 10.4049/jimmunol.1800384

Figure Lengend Snippet: a. Screening of anti-human P mAbs by ELISA. Culture supernatant from 4 hybridoma clones, mAb 19.1, 22.1, 25 and 30, showed strong binding to plate-immobilized hP, where supernatant from a negative mAb clone 27.1 showed no binding. b. Inhibition of LPS-induced AP complement activation by affinity purified mAb 19.1, 22.1, 25 and 30. All 4 clones of mAbs effectively inhibited AP complement activation when added to 10% normal human serum (NHS) at a final concentration of 1μg/ml. A sample with EDTA added (EDTA) served as a positive control for inhibition. A sample with no mAb added (NHS) served as the baseline AP complement activation. OD450 value represents ELISA reading of relative C3 fragments deposition (i.e. activated and plate-bound C3). c. Western blot confirming that mAb clone 19.1 specifically recognizes purified properdin and properdin in NHS. Lane 1: 20 ng of purified human properdin; Lane 2: 1μl of NHS separated on non-reducing SDS-PAGE. mAb 19.1 was used at 2 μg/ml in Western blotting. d. Further functional characterization of mAb clone 19.1 showing it concentration-dependently inhibiting LPS-induced AP complement activity in 50% NHS. NHS was diluted 1:1 (i.e. to 50%) in EGTA-Mg+2 supplemented GVB++ buffer and then pretreated with or without mAb (1, 2.5 and 5μg/ml) for 1hr at 4°C before adding to LPS-coated plates. It completely blocked AP complement activity at 5μg/ml in this assay. EDTA serum (NHS+EDTA) was used as a positive control for complement inhibition. e, f. Human erythrocytes were lysed by 50% NHS in the presence of human factor H SCR19-20 and anti-DAF antibody (7.5 μg/ml, panel e) or anti-DAF and anti-CD59 antibodies (10 μg/ml for both, panel f). Lysis was prevented by EDTA or mAb 19.1 (5 μg/ml for panel e and as indicated in panel f). Assays were performed in triplicates and percent lysis was normalized to hypotonic lysis (100%) with distilled water (DDW). All data are representative of at least three independent experiments and values are expressed as mean (SD) of at least 3 replicate assays per data point. * p<0.001 compared with sample without mAb 19.1 treatment (NHS). One-way ANOVA.

Article Snippet: Plates were then washed 3 times with PBS-T and captured human P was detected using a biotinylated goat anti-human P antibody (CompTech, Cat # A239) and standard HRP-based ELISA detection system.

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, Binding Assay, Inhibition, Activation Assay, Affinity Purification, Concentration Assay, Positive Control, Western Blot, Purification, SDS Page, Functional Assay, Activity Assay, Lysis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Antibody inhibition of properdin prevents complement-mediated intravascular and extravascular hemolysis 1

doi: 10.4049/jimmunol.1800384

Figure Lengend Snippet: a. Human properdin cDNA construct used for transgenic mouse generation. The cDNA cassette is composed of the chicken β-actin promoter with CVM-IE enhancer, human P cDNA and the rabbit β -globin polyA tail for stable expression of the cDNA in eukaryotic cells. b. Genotyping of hP transgenic mice by PCR using hP-specific primers and tail DNA. Of 40 mice screened, five transgene-positive founders were identified by the presence of a PCR fragment of approximate 800 bp (#15, #20, #24, #27 and #32). c. Human P protein was detected by ELISA in the sera of the 5 transgene-positive mice but not in a transgene-negative mouse (#29). Normal mouse serum (NMS, i.e. serum from wild-type mouse) and serum from P−/− mouse were also negative for hP whereas normal human serum (NHS) was positive for hP. * p<0.0001, NS: non-significant. One-way ANOVA comparing with NMS or P−/− mouse serum. d. Transgenically expressed hP restored AP complement activity to P−/− mice. In contrast to P−/− mouse serum which showed no LPS-dependent AP complement activity, sera from two P−/− mice with hP transgene expression (HuPTg1 and HuPTg2) showed similar AP complement activity to that of a WT mouse. Data in panel c and d are representative of 2-3 independent experiments. Mean (SD) of triplicate assays (c) or average of duplicate assays (d) are shown.

Article Snippet: Plates were then washed 3 times with PBS-T and captured human P was detected using a biotinylated goat anti-human P antibody (CompTech, Cat # A239) and standard HRP-based ELISA detection system.

Techniques: Construct, Transgenic Assay, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: PLoS ONE

Article Title: The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region

doi: 10.1371/journal.pone.0253260

Figure Lengend Snippet: (A) Competition assay. Serial 3-fold dilutions of either WT or 8-site mutated sFasL were mixed with biotinylated WT sFasL (70 ng/ml) and incubated for 2 hours in 96-well plates pre-coated with recombinant human soluble Fas (250 ng/ml). Biotin was detected using HRP conjugated streptavidin; (B) Fas binding assay. Serial 3-fold dilutions of wild type (WT) or mut-sFasL were incubated for 2 hours in wells coated with recombinant human soluble Fas, 5 μg/ml. The sFasL bound to Fas was detected using a polyclonal anti-human FasL antibody. The mut sFasL binding curve was shifted to the left. n = 3. (C) Jurkat cells were incubated in 96-well plates at a concentration of 1 x 10 4 cells/well. Different molar ratios of the Fas-activating antibody CH-11 and mut-sFasL were added, and caspase 3/7 activity measured after 5 hours. The mut-sFasL did not inhibit the activity of CH11. Data represent the results from three separate experiments, each done in duplicate, and were analyzed by 2-way ANOVA with Sidak’s post hoc analysis; (A), WT-sFasL compared to mut-sFasL at the same concentrations; (B), comparisons made to no competitor condition for each molecule; (C) Comparisons made to unmixed antibody (Ab). Each dot represents individual data, lines represent means ± SD. * = P < 0.05; ** = P<0.01, ***P<0.001.

Article Snippet: For the Fas-FasL binding assay, we used recombinant human soluble Fas receptors (PeproTech, Rocky Hill, NJ) and biotinylated goat anti-human FasL polyclonal antibodies (PeproTech Cat# 500-P184bt-50ug, RRID:AB_148184).

Techniques: Competitive Binding Assay, Incubation, Recombinant, Binding Assay, Concentration Assay, Activity Assay

Journal: PLoS ONE

Article Title: The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region

doi: 10.1371/journal.pone.0253260

Figure Lengend Snippet: Serial 3-fold dilutions of FLAG tagged mut-sFasL were mixed with biotinylated WT sFasL (60 ng). The mixtures were incubated at room temperature for 15 minutes and applied to streptavidin-coated wells. FLAG tags were detected with HRP anti-FLAG mAb. There was a dose-dependent increase in FLAG signal in wells incubated with the biotinylated WT sFasL and the mut-sFasL-FLAG mixture, indicating formation of complexes of WT sFasL and mut-sFasL. The mut-sFasL-FLAG without biotin did not bind to the streptavidin-coated plates. Data shown as ratio of the absorbance at 450 nm of each experimental condition to that of media only. Data were generated from three separate experiments, each done in duplicate, and were analyzed by 2-way ANOVA with Dunnet’s post hoc analysis; comparisons were made to 0 ng/mL for each condition. Each dot represents individual data, lines represent means ± SD. * = P < 0.05; ** = P<0.01, ***P<0.001.

Article Snippet: For the Fas-FasL binding assay, we used recombinant human soluble Fas receptors (PeproTech, Rocky Hill, NJ) and biotinylated goat anti-human FasL polyclonal antibodies (PeproTech Cat# 500-P184bt-50ug, RRID:AB_148184).

Techniques: Incubation, Generated