Journal:

Article Title: Melanoma inhibitor of apoptosis protein (ML-IAP) is a target for immune-mediated tumor destruction

doi: 10.1073/pnas.0530311100

Figure Lengend Snippet: Vaccination-augmented anti-ML-IAP antibody titers and induced isotype switching. (A) Pan-IgG. (B) IgG4. The ELISA was performed with recombinant GST-ML-IAP protein and K030 sera diluted 1:10,000 for the pan-IgG determinations and 1:100 for the IgG4 measurements. Small arrows denote vaccinations. The peak pan-IgG and IgG4 levels were 4-fold and 10-fold higher than the respective values at study entry. K030 sera also recognized GST-ML-IAP in Western analysis. Reactivity to Candida antigens was not consistently affected by therapy (data not shown).

Article Snippet: A polyclonal goat anti-human pan IgG (Jackson ImmunoResearch, 1:1,000 dilution), monoclonal mouse anti-IgG1 (Zymed, 1:500 dilution), or monoclonal mouse anti-IgG4 (Southern Biotechnology Associates, 1:500 dilution) conjugated to alkaline phosphatase in 2% NFDM/PBS was added at room temperature for 1 h, and the plate was developed with p -nitrophenyl phosphate (Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Western Blot

Journal: Scientific Reports

Article Title: Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient

doi: 10.1038/s41598-017-14350-8

Figure Lengend Snippet: Overview of IgG1-131 & IgG4-131 cloning strategy and production. The expression cassettes for the production of (a) VH-CH and (b) Vκ-Cκ segments of the mAb 131 in IgG format are shown. (c) Isotype specificities of the recombinant antibodies along with control mAbs were analyzed by dot blot using isotype specific HRP-conjugated antibodies.

Article Snippet: Purified mAbs IgG1-131, IgG4-131, as well as mAbs IgG1-637 & IgG4-637 as controls, were blotted on nitrocellulose membranes (Biorad, Cat No. 162-0116) for 1 h at 37 °C, blocked with 5% non-fat dry milk in 1x PBS for 1 h at 37 °C, washed with PBS-T (containing 0.05% Tween20) and incubated with HRP-conjugated goat F(ab’) 2 anti-human IgG-Fcγ (Jackson Immuno-Research, Cat No. 109-036-008; diluted 1:2500) or mouse anti-human IgG1 (Invitrogen, Cat No. 05-3320; diluted 1:2500) or mouse anti-human IgG4 (AbD serotec, Cat No. MCA2098P; diluted 1:2500) secondary antibody.

Techniques: Cloning, Expressing, Recombinant, Control, Dot Blot

Journal: Scientific Reports

Article Title: Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient

doi: 10.1038/s41598-017-14350-8

Figure Lengend Snippet: Recombinant IgG1-131 binds fetal AChR and γ-subunit with high specificity and apparent affinity. (a) The α, β and γ-subunits of the human muscle AChR were resolved on SDS-PAGE (shown in upper panel) and specificity of IgG1-131 for γ - subunit was analyzed by Western blot (lower panel). Full image of the SDS-PAGE and Western blot are shown in Supplementary Fig. . (b) ELISA analysis of IgG1-131 (0.01 nM – 10 nM) binding to the α, β and γ-subunit of the AChR. (c) Radio-immunoprecipitation of IgG1-131 using fetal and adult AChR extracted from TE671 and CN21 cell membranes respectively. (d) Cross-reactivity of IgG1-131 against rat and mouse muscle AChR, and the Torpedo electric organ AChR was analyzed by radio-immunoprecipitation assay. (e) The specificities of IgG1-131 and IgG4-131 were tested against fetal (TE671 membrane extract) and adult AChR (CN21 membrane extract). IgG1-637 and IgG4-637 were used as a reference in both tests. (f) 125 I-α-BT blocking was measured as percentage inhibition in the 125 I-α-BT binding to AChRs after co-incubating the individual antibody/AMC serum and 125 I-α-BT labelled AChR. The results are from two independent experiments. (g) Analysis of competition between IgG1-131 and IgG1-637. IgG1-131 (100 nM) when co-incubated with IgG1-637 (25 nM), reduced the amount of 125 I-α-BT co-precipitated with fetal AChR compared to 25 nM IgG1-637 alone. (h) Schematic representation of the binding effects of mAb 131 and mAb 637. The mAb 637 binds to the two designated α-subunits of the fetal AChR, allowing simultaneous binding of two α-BT molecules. However, in the presence of competing/blocking mAb 131, one of the α-BT molecules and mAb 637 at the α/γ interface gets displaced by mAb 131.

Article Snippet: Purified mAbs IgG1-131, IgG4-131, as well as mAbs IgG1-637 & IgG4-637 as controls, were blotted on nitrocellulose membranes (Biorad, Cat No. 162-0116) for 1 h at 37 °C, blocked with 5% non-fat dry milk in 1x PBS for 1 h at 37 °C, washed with PBS-T (containing 0.05% Tween20) and incubated with HRP-conjugated goat F(ab’) 2 anti-human IgG-Fcγ (Jackson Immuno-Research, Cat No. 109-036-008; diluted 1:2500) or mouse anti-human IgG1 (Invitrogen, Cat No. 05-3320; diluted 1:2500) or mouse anti-human IgG4 (AbD serotec, Cat No. MCA2098P; diluted 1:2500) secondary antibody.

Techniques: Recombinant, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Radio Immunoprecipitation, Membrane, Blocking Assay, Inhibition, Incubation

Journal: Scientific Reports

Article Title: Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient

doi: 10.1038/s41598-017-14350-8

Figure Lengend Snippet: IgG1-131 binds to human RMS cell lines. (a) Binding of IgG1-131 and IgG1-637 to cell surface fetal AChR on different RMS cell lines was analyzed by immunofluorescence. Binding of antibody was detected by goat anti-human IgG-Alexa 488 antibody (green) and Hoechst was used to stain the nuclei. HEK293 cells were used as negative control for each antibody. Representative images at 20x magnification are shown, n = 2. Scale bar = 50 µm. (b) Analysis of 125 I-α-BT bound to cell surface AChR on RMS cell lines after incubation with IgG1-131 or IgG1 637. Individual bars correspond to the mean ± SD of three replicates tested for each condition. Results are representative of 3 independent experiments.

Article Snippet: Purified mAbs IgG1-131, IgG4-131, as well as mAbs IgG1-637 & IgG4-637 as controls, were blotted on nitrocellulose membranes (Biorad, Cat No. 162-0116) for 1 h at 37 °C, blocked with 5% non-fat dry milk in 1x PBS for 1 h at 37 °C, washed with PBS-T (containing 0.05% Tween20) and incubated with HRP-conjugated goat F(ab’) 2 anti-human IgG-Fcγ (Jackson Immuno-Research, Cat No. 109-036-008; diluted 1:2500) or mouse anti-human IgG1 (Invitrogen, Cat No. 05-3320; diluted 1:2500) or mouse anti-human IgG4 (AbD serotec, Cat No. MCA2098P; diluted 1:2500) secondary antibody.

Techniques: Binding Assay, Immunofluorescence, Staining, Negative Control, Incubation

Journal: Scientific Reports

Article Title: Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient

doi: 10.1038/s41598-017-14350-8

Figure Lengend Snippet: IgG1-131 does not lead to modulation of fetal AChR on TE671 cells, but partly displaces 125 I-α-BT. The effect of (a) IgG1-637, (b) IgG4-637, (c) IgG1-131 and (d) IgG4-131 on the 125 I-α-BT binding to TE671 (fetal AChR) and CN21 (adult AChR) cells is shown as normalized cell bound radioactivity at varying molar concentrations of the antibodies. Cells were incubated with antibodies for 3 h and subsequently labelled with 125 I-α-BT. Each data point corresponds to the mean ± SD of three replicates. (e) Effect of IgG1-131 and IgG1-637 on 125 I-α-BT binding to TE671 cells at different time points (1, 2 and 3 h). For each time point, cells cultured in medium without any IgG were used as controls. Unspecific binding of 125 I-α-BT was measured by pre-incubation of cells with an excess of unlabeled α-BT (25 nM). Data points of 1, 2 and 3 hours incubation were pooled for this condition. (f) Schematic representation of the Fab arm-exchange reaction under reducing conditions between IgG4-131 (VH and VL domains shown in red and CH in black color) and IgG4 from normal human serum (IgG4-NHS; VH and VL domains shown in blue and CH in grey color) to yield two bispecific IgG4 molecules. (g) Purity of IgG4 isolated from normal human serum (NHS) was validated by dot blot assay along with control mAbs. (h) Competition experiments using monovalent FAE IgG4-131 and IgG4-637. The loss of the cell bound radioactivity due to the IgG1-131 incubation was not significantly affected by an excess FAE IgG4-131. In contrast, FAE IgG4-637 completely counteracted the effect of IgG1-637. Data are pooled from 3 experiments with representative groups.

Article Snippet: Purified mAbs IgG1-131, IgG4-131, as well as mAbs IgG1-637 & IgG4-637 as controls, were blotted on nitrocellulose membranes (Biorad, Cat No. 162-0116) for 1 h at 37 °C, blocked with 5% non-fat dry milk in 1x PBS for 1 h at 37 °C, washed with PBS-T (containing 0.05% Tween20) and incubated with HRP-conjugated goat F(ab’) 2 anti-human IgG-Fcγ (Jackson Immuno-Research, Cat No. 109-036-008; diluted 1:2500) or mouse anti-human IgG1 (Invitrogen, Cat No. 05-3320; diluted 1:2500) or mouse anti-human IgG4 (AbD serotec, Cat No. MCA2098P; diluted 1:2500) secondary antibody.

Techniques: Binding Assay, Radioactivity, Incubation, Cell Culture, Isolation, Dot Blot, Control

Journal: Scientific Reports

Article Title: Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient

doi: 10.1038/s41598-017-14350-8

Figure Lengend Snippet: IgG1-131 does not inhibit fetal AChR function. (a) AChR currents (mean ± SEM) in individual cells (n = 6) were measured during the continuous perfusion with control solution (without IgG1-131) and with test solution (application of 5 μg/mL purified IgG1-131 for 2.5 min). (b) Repetitive AChR current measurements in an individual cell continuously perfused with 10 mL of test solution. Data points are the means ( ± SEM) of a number of measurements (control solution: n = 10, test solution: n = 6 at 0.5 min, n = 6 at 8 min, n = 6 at 15 min and n = 3 at 22 min). AChR currents were normalized to the cell capacitance and expressed as pA/pF.

Article Snippet: Purified mAbs IgG1-131, IgG4-131, as well as mAbs IgG1-637 & IgG4-637 as controls, were blotted on nitrocellulose membranes (Biorad, Cat No. 162-0116) for 1 h at 37 °C, blocked with 5% non-fat dry milk in 1x PBS for 1 h at 37 °C, washed with PBS-T (containing 0.05% Tween20) and incubated with HRP-conjugated goat F(ab’) 2 anti-human IgG-Fcγ (Jackson Immuno-Research, Cat No. 109-036-008; diluted 1:2500) or mouse anti-human IgG1 (Invitrogen, Cat No. 05-3320; diluted 1:2500) or mouse anti-human IgG4 (AbD serotec, Cat No. MCA2098P; diluted 1:2500) secondary antibody.

Techniques: Control, Purification