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

Journal: Virologica Sinica

Article Title: Omicron variants breakthrough infection elicited higher specific memory immunity than third dose booster in healthy vaccinees.

doi: 10.1016/j.virs.2022.12.008

Figure Lengend Snippet: Fig. 1. Comparative analysis of anti-ancestral-spike-trimer-IgG. Serum were collected from vaccinated healthy volunteers (HVs) before third booster, at median of 33 days post inactivated vaccine (InV) booster and 30.5 days post Ad5-nCoV booster, and Omicron variants breakthrough infected patients (OBIPs) at median of 32 days post infection. A Anti-spike-trimer-IgG titer in InV boosted HVs, Ad5-nCoV boosted HVs, and OBIPs were determined by ELISA. B–G Comparative analysis of SARS- CoV-2 ancestral strain spike-trimer specific IgG subclass. Anti-spike-trimer-IgG1 (B), anti-Spike-trimer-IgG2 (C), anti-Spike-trimer-IgG3 (D), and anti-spike-trimer-IgG4 (E) titer in InV boosted HVs, Ad5-nCoV boosted HVs, and OBIPs were determined by ELISA. F The ratio of spike-trimer specific IgG1/IgG4 in InV boosted HVs, Ad5- nCoV boosted HVs, and OBIPs. G The ratio of spike-trimer specific IgG1þIgG3/IgG4þIgG2 in InV boosted HVs, Ad5-nCoV boosted HVs, and OBIPs. Data presented as geometric mean titers (GMT) and 95% confidence interval. Dotted line: the detection limits. Statistical analyses were performed by Mann-Whitney U test.

Article Snippet: After blocking, 100 μL 3-fold diluted plasma (initially dilute for IgG, IgG1, IgG2 and IgG3:1:20, IgG4:1:10) was added in each well, and incubated at 37 C for 1 h. After thorough washing, 100 μL HRP labeled secondary antibodies against human IgG (Cat#: 2010-05, 1:10,000, Southern Biotech, USA), IgG1 (Cat#: 9054-05, 1:4000, Southern Biotech, USA), IgG2 (Cat#: 9060- 05, 1:2000, Southern Biotech, USA), IgG3 (Cat#: 9210-05, 1:4000, Southern biotech, USA), and IgG4 (Cat#: 9200-05, 1:4000, Southern Biotech, USA) was added in each well.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Virologica Sinica

Article Title: Omicron variants breakthrough infection elicited higher specific memory immunity than third dose booster in healthy vaccinees.

doi: 10.1016/j.virs.2022.12.008

Figure Lengend Snippet: Fig. 2. Comparative analysis of anti-RBD antibody against SARS-CoV-2. Serum were collected vaccinated healthy volunteers (HVs) before third booster, at median of 33 days post InV booster and 30.5 days post Ad5-nCoV booster, and OBIPs at median of 32 days post infection. Anti-ancestral-receptor binding domain (RBD)-IgG (A), anti-Delta-RBD-IgG (B), and anti-Omicron-RBD-IgG (C) titer in pre-boosted HVs, InV boosted HVs, Ad5-nCoV boosted HVs, and OBIPs were determined by ELISA. D Paired analysis of anti-ancestral-RBD-, anti-Delta-RBD-, and anti-Omicron-RBD-IgG titer in pre-boosted HVs, InV boosted HVs, Ad5-nCoV boosted HVs, and OBIPs. Data presented as geometric mean titers (GMT) and 95% confidence interval. Dotted line: the detection limits. Statistical analyses were performed by Mann-Whitney U test.

Article Snippet: After blocking, 100 μL 3-fold diluted plasma (initially dilute for IgG, IgG1, IgG2 and IgG3:1:20, IgG4:1:10) was added in each well, and incubated at 37 C for 1 h. After thorough washing, 100 μL HRP labeled secondary antibodies against human IgG (Cat#: 2010-05, 1:10,000, Southern Biotech, USA), IgG1 (Cat#: 9054-05, 1:4000, Southern Biotech, USA), IgG2 (Cat#: 9060- 05, 1:2000, Southern Biotech, USA), IgG3 (Cat#: 9210-05, 1:4000, Southern biotech, USA), and IgG4 (Cat#: 9200-05, 1:4000, Southern Biotech, USA) was added in each well.

Techniques: Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal of Clinical Laboratory Analysis

Article Title: Evaluation of autoantibodies and immunoglobulin G subclasses in women with suspected macroprolactinemia

doi: 10.1002/jcla.23456

Figure Lengend Snippet: IgG subclass distributions in Group 1 and Group 2 patients

Article Snippet: The membranes were then probed with the anti‐human IgG1 antibody (ThermoFisher, A10648, USA), anti‐human IgG2 antibody (SouthernBiotech, 9070‐01, USA), anti‐human IgG3 antibody (SouthernBiotech, 9210‐01), or anti‐human IgG4 antibody (SouthernBiotech, 9200‐01) separately, followed by incubation with the IRDyeTM secondary antibodies (1:20 000).

Techniques:

Journal: Journal of Clinical Laboratory Analysis

Article Title: Evaluation of autoantibodies and immunoglobulin G subclasses in women with suspected macroprolactinemia

doi: 10.1002/jcla.23456

Figure Lengend Snippet: Immunoprecipitation with the serum samples of the patients suspected for macroprolactinemia. A, Relative levels of PRL‐specific IgG subclasses associated with PRL‐IgG complexes in patients with anti‐PRL autoantibodies are shown in bar graph. Data are expressed as mean ± standard deviation. B, Representative Western blots with the patients’ serum samples from Group 1 (#117, 159, 196, 229) and Group 2 (#120, 262, 302, 322). Lane 1: positive control loaded with pure IgG1‐IgG4 proteins

Article Snippet: The membranes were then probed with the anti‐human IgG1 antibody (ThermoFisher, A10648, USA), anti‐human IgG2 antibody (SouthernBiotech, 9070‐01, USA), anti‐human IgG3 antibody (SouthernBiotech, 9210‐01), or anti‐human IgG4 antibody (SouthernBiotech, 9200‐01) separately, followed by incubation with the IRDyeTM secondary antibodies (1:20 000).

Techniques: Immunoprecipitation, Standard Deviation, Western Blot, Positive Control

Journal: Protein engineering, design & selection : PEDS

Article Title: Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.

doi: 10.1093/protein/gzu010

Figure Lengend Snippet: Fig. 2. Demonstration of A9 as an exosite TACE inhibitor: (A) A9 IgG is a non-competitive inhibitor of TACE catalytic-cleft (active site). The dependence of the peptidolytic activity of TACE ectodomain on the concentration of the fluorogenic substrate (methoxycoumarinyl acetyl—Lys-Pro-Leu-Gly-Leu-dinitrophenyl diaminopropionyl-Ala-Arg-NH2) in the presence of A9 IgG at concentrations of 0–250 nM is shown (left panel). For each A9 IgG concentration, the Kapp m and Vapp max were estimated by fitting the data to equation (1). The Vapp max values were plotted against the A9 IgG concentration, and the data were fit to equation (2), expected to apply for non-competitive inhibition. The fit yielded a Ki value of 83.5+0.2 nM (right panel). (B) A9 IgG causes the catalytic-cleft conformational change affected the binding of the low-molecular-mass hydroxamate inhibitors of metalloproteinases CT1746. Emission spectra of CT1746 (þ), TACE alone (B) or CT1746–TACE complex (V) were collected with an excitation wavelength of 230 nm, and emission spectra were scanned from 280 to 400 nm. In parallel, samples of CT1746 in addition of TACE in the presence of titrated A9 IgG (5, 0.5, 0.05 and 0 mM) and human IgG control were recorded. (C) Comparison of SPR plot for A9 IgG to TACE alone and CT1746–TACE complex. The dissociation constant of A9 IgG to TACE was reduced approximately to half in the presence of CT1746.

Article Snippet: TACE cell-surface shedding assays For all shedding assays, 1 105 cells/well (in 320 ml medium) were plated in a 48-well plate for 18 h, washed one time with serum free media and pre-incubated with either A9 IgG mutants, CT1746 or control human plasma IgG (R&D Systems) (diluted in serum free media) for 1 h. Each well was stimulated/ treated with either 100 ng/ml phorbol 12-myristate 13-acetate (PMA), 2.5 mM ionomycin or 50 nM gemcitabine, and supernatants were harvested after 1–24 h (depending on the cell lines and treatments).

Techniques: Activity Assay, Concentration Assay, Inhibition, Binding Assay, Control, Comparison

Journal: Protein engineering, design & selection : PEDS

Article Title: Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.

doi: 10.1093/protein/gzu010

Figure Lengend Snippet: Fig. 3. Enhancement of A9 mutants: (A) human and mouse TACE ectodomain proteolytic activity of the top eight A9 mutants (IC50 values) were measured in a QF peptide cleavage assay. (B) Example of SPR plot for lead A9 IgG mutant—B8 (left panel). The dissociation constant of all top eight A9 IgG mutants to TACE ectodomain was determined by BIAcore technology on a CM5 chip using the human antibody capture kit which revealed the sub-nanomolar dissociation constant KD (right panel).

Article Snippet: TACE cell-surface shedding assays For all shedding assays, 1 105 cells/well (in 320 ml medium) were plated in a 48-well plate for 18 h, washed one time with serum free media and pre-incubated with either A9 IgG mutants, CT1746 or control human plasma IgG (R&D Systems) (diluted in serum free media) for 1 h. Each well was stimulated/ treated with either 100 ng/ml phorbol 12-myristate 13-acetate (PMA), 2.5 mM ionomycin or 50 nM gemcitabine, and supernatants were harvested after 1–24 h (depending on the cell lines and treatments).

Techniques: Activity Assay, Cleavage Assay, Mutagenesis

Journal: Protein engineering, design & selection : PEDS

Article Title: Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.

doi: 10.1093/protein/gzu010

Figure Lengend Snippet: Fig. 5. Lead A9 IgG mutant—B8 inhibition in a gemcitabine-treated PDAC in vitro model derived from KPC mice: (A) DT8082 cell line from KPC mice was treated either alone or with gemcitabine. At 16 h, western blot analysis of TACE was performed on their cell lysates. Cells lysates were subjected to SDS–PAGE, transferred on to nitrocellulose membranes and probed with a relevant antibody. (B) DT8082 cell line was treated either alone, with gemcitabine or with gemcitabine þ IgG control/B8/CT1746 for 16 h. TACE substrate AREG was quantified from conditioned media by sandwich ELISA.

Article Snippet: TACE cell-surface shedding assays For all shedding assays, 1 105 cells/well (in 320 ml medium) were plated in a 48-well plate for 18 h, washed one time with serum free media and pre-incubated with either A9 IgG mutants, CT1746 or control human plasma IgG (R&D Systems) (diluted in serum free media) for 1 h. Each well was stimulated/ treated with either 100 ng/ml phorbol 12-myristate 13-acetate (PMA), 2.5 mM ionomycin or 50 nM gemcitabine, and supernatants were harvested after 1–24 h (depending on the cell lines and treatments).

Techniques: Mutagenesis, Inhibition, In Vitro, Derivative Assay, Western Blot, SDS Page, Control, Sandwich ELISA

Journal: Vaccines

Article Title: Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

doi: 10.3390/vaccines13020140

Figure Lengend Snippet: Characteristics of serum antibodies to each HA region induced by seasonal influenza vaccination. Total IgG, IgG1, and IgG2 antibodies against vaccine strains HA head-specific, H1 stem-specific, and LAH epitopes specific to vaccine strains H1 and H3 were quantified by ELISA using pre- and post-vaccination sera from seasonal influenza vaccines. Quantification was performed using ELISA. ( A ) Total IgG antibody titers against HA head-specific, H1 stem-specific, and LAH epitopes specific to vaccine strains H1 and H3 before and after vaccination. ( B ) IgG1 and IgG2 antibody titers against H1 HA head-specific before and after vaccination. ( C ) IgG1 and IgG2 antibody titers against H3 HA head-specific antibodies before and after vaccination. Total IgG, IgG1, and IgG2 antibody titers against the H1 stem before and after vaccination. ( D ) IgG1 and IgG2 antibody titers against H1 stem cells before and after vaccination. Antibody titers were quantified using standard antibodies, and the amounts are shown. Each circle represents the results for an individual participant; lines represent median values. The fold change is shown above the graph as the median antibody titer in the post-vaccination group divided by that in the pre-vaccination group. The data were analyzed using the Wilcoxon matched-pair signed-rank test. Significance is indicated by the following symbols: *** p < 0.001, and **** p < 0.0001.

Article Snippet: Next, the plates were washed and incubated for 1 h with various secondary anti-human IgG antibodies, including mouse anti-Human IgG Fc-BIOT (SouthernBiotech, USA), mouse anti-Human IgG1 Fc secondary antibody-HRP (Thermo Fisher Scientific, Waltham, MA, USA), mouse Anti-Human IgG2 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA), mouse Anti-Human IgG3 Hinge-BIOT (SouthernBiotech, Birmingham, AL, USA), or mouse Anti-Human IgG4 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Vaccines

Journal: Vaccines

Article Title: Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

doi: 10.3390/vaccines13020140

Figure Lengend Snippet: Cross-reactive neutralizing antibody activity in serum induced by current seasonal influenza vaccination. Post-vaccination sera were subjected to a microneutralization assay using ten different influenza viruses and representative data from two independent experiments. The results obtained were scored as follows: 0 for 20 and below, 1 for 21 to 80, 2 for 81 to 640, 3 for 641 to 1280, and 4 for 1280 and above. Neutralization breath scores were calculated by summing the respective scores for groups 1, 2, and group 1 + 2. Neutralization breath scores were calculated by summing the scores of groups 1, 2, and group 1 + 2. ( A ) Correlation between neutralization group 1 breath score and total IgG of H1, H3 HA head-specific and H1 stem-specific. ( B ) Correlation of neutralization group 2 breath score with total IgG for H1, H3 HA head-specific, and H1 stem-specific. Data were statistically analyzed using Spearman’s rank correlation coefficients. Lines indicate correlations determined by linear regression analysis ( n = 50 for graphs of H1 and H3 HA head-specific antibodies and n = 36 for graphs of H1 stem-specific antibodies).

Article Snippet: Next, the plates were washed and incubated for 1 h with various secondary anti-human IgG antibodies, including mouse anti-Human IgG Fc-BIOT (SouthernBiotech, USA), mouse anti-Human IgG1 Fc secondary antibody-HRP (Thermo Fisher Scientific, Waltham, MA, USA), mouse Anti-Human IgG2 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA), mouse Anti-Human IgG3 Hinge-BIOT (SouthernBiotech, Birmingham, AL, USA), or mouse Anti-Human IgG4 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA).

Techniques: Activity Assay, Microneutralization Assay, Neutralization

Journal: Vaccines

Article Title: Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

doi: 10.3390/vaccines13020140

Figure Lengend Snippet: Cross-reactive ADCC activity induced by current seasonal influenza vaccination in serum. ADCC assays were performed on post-vaccination sera using 10 different influenza viruses, representative data from two independent experiments. The results obtained were scored as follows: 0 for 10 or less, 1 for 11 to 100, 2 for 101 to 500, 3 for 501 to 1000, and 4 for 1000 or more. Breath scores were calculated by summing the scores of group 1, group 2, and group 1 + 2. ( A ) Correlation between the ADCC group 1 breath score and H1 HA head-specific total IgG, IgG1, and IgG2 levels. ( B ) Correlation between the ADCC group 2 breath score and H3 HA head-specific total IgG and IgG1 levels. ( C ) Correlation between ADCC group 1 breath score and H1 stem-specific total IgG and IgG1 levels. Data were statistically analyzed using Spearman’s rank correlation coefficients. Lines indicate correlations determined by linear regression analysis ( n = 50 for graphs of H1 and H3 HA head-specific antibodies and n = 36 for graphs of H1 stem-specific antibodies).

Article Snippet: Next, the plates were washed and incubated for 1 h with various secondary anti-human IgG antibodies, including mouse anti-Human IgG Fc-BIOT (SouthernBiotech, USA), mouse anti-Human IgG1 Fc secondary antibody-HRP (Thermo Fisher Scientific, Waltham, MA, USA), mouse Anti-Human IgG2 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA), mouse Anti-Human IgG3 Hinge-BIOT (SouthernBiotech, Birmingham, AL, USA), or mouse Anti-Human IgG4 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA).

Techniques: Activity Assay

Journal: Vaccines

Article Title: Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

doi: 10.3390/vaccines13020140

Figure Lengend Snippet: Cross-reactive ADCP activity induced by current seasonal influenza vaccination in serum. ADCP assays were performed on post-vaccination plasma with 10 different influenza viruses, and representative data were obtained from two independent experiments. The results were scored as follows: 0, 10 and below; 1, 11–100; 2, 101–500; 3, 501–1000; and 4, 1000 and above. Breath scores were calculated by summing the respective scores for groups 1, 2, and 1 + 2. ( A ) Correlation between the ADCP group 1 breath score and H1 HA head-specific total IgG, IgG1, and IgG2 levels. ( B ) Correlation between ADCP group 2 breath score and H3 HA head-specific total IgG and IgG1 levels. ( C ) Correlation between the ADCP group 1 breath score and H1 stem-specific total IgG and IgG1. Data were statistically analyzed using Spearman’s rank correlation coefficients. Lines indicate correlations determined by linear regression analysis ( n = 50 for graphs of H1 and H3 HA head-specific antibodies and n = 36 for graphs of H1 stem-specific antibodies).

Article Snippet: Next, the plates were washed and incubated for 1 h with various secondary anti-human IgG antibodies, including mouse anti-Human IgG Fc-BIOT (SouthernBiotech, USA), mouse anti-Human IgG1 Fc secondary antibody-HRP (Thermo Fisher Scientific, Waltham, MA, USA), mouse Anti-Human IgG2 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA), mouse Anti-Human IgG3 Hinge-BIOT (SouthernBiotech, Birmingham, AL, USA), or mouse Anti-Human IgG4 Fc-BIOT (SouthernBiotech, Birmingham, AL, USA).

Techniques: Activity Assay, Clinical Proteomics

Journal: Molecular Therapy

Article Title: C3d-Targeted factor H inhibits tissue complement in disease models and reduces glomerular injury without affecting circulating complement

doi: 10.1016/j.ymthe.2024.02.001

Figure Lengend Snippet: C3d-mAb-2fH binding affinity rises with increased C3d density

Article Snippet: Rodent C3d was stained using a human anti-C3d IgG4 (clone 3d8b, ADX-086) followed by a secondary Alexa Fluor 647-conjugated mouse anti-human IgG4 pFc′ (Southern Biotech 9190-31 clone HP6023) or Alexa Fluor 488-conjugated mouse anti-human IgG4 pFc′ (Southern Biotech 9190-30 clone HP6023).

Techniques: Binding Assay

Journal: Molecular Therapy

Article Title: C3d-Targeted factor H inhibits tissue complement in disease models and reduces glomerular injury without affecting circulating complement

doi: 10.1016/j.ymthe.2024.02.001

Figure Lengend Snippet: In vitro complement inhibition by C3d-mAb-fH fusion proteins

Article Snippet: Rodent C3d was stained using a human anti-C3d IgG4 (clone 3d8b, ADX-086) followed by a secondary Alexa Fluor 647-conjugated mouse anti-human IgG4 pFc′ (Southern Biotech 9190-31 clone HP6023) or Alexa Fluor 488-conjugated mouse anti-human IgG4 pFc′ (Southern Biotech 9190-30 clone HP6023).

Techniques: In Vitro, Inhibition, Activity Assay

Journal: Nature Communications

Article Title: Divergent trajectories of antiviral memory after SARS-CoV-2 infection

doi: 10.1038/s41467-022-28898-1

Figure Lengend Snippet: Humoral immune responses were assessed in acute and convalescent by binding antibody ELISA for total IgG specific to the a Spike glycoprotein and b Nucleocapsid, quantification of c IgG memory B cells specific to the spike glycoprotein, and d pseudoneutralisation antibody titres. A two-tailed Wilcoxon rank-sum test was used to compare between study time points. The boxplots all display the median values with the first and third quartile, and the whiskers represent the highest and lowest values no more than 1.5 times the interquartile range from the corresponding hinge. A generalised additive mixed model (GAMM) by restricted maximum likelihood—right-hand plots—was used to fit the immunological measures (log10 transformed) taken at multiple study time points, using Gaussian process smooth term. The GAMM plots the ribbon represents the 95% confidence interval around the fitted value. Disease severity group was included in the GAMM as a linear predictor and a participant identifier was included as a random effect. See Table for number of individuals evaluated per assay.

Article Snippet: For detection of anti-spike IgG2 and IgG4 steps modified as follows: (1) Plates were additionally coated with commercially available human immunoglobulin control (recombinant human IgG2 lambda or recombinant human IgG4 lambda (Bio-Rad)) to serve as internal controls, (2) Mouse anti-human IgG2 Fd-AP or mouse anti-human IgG4 Fc-AP (Southern Biotech) were used, and (3) Optical density at 405 nm was measured using an ELx808 absorbance reader (BioTek) until the immunoglobulin control reached a specified OD405.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Transformation Assay

Journal: Nature Communications

Article Title: Divergent trajectories of antiviral memory after SARS-CoV-2 infection

doi: 10.1038/s41467-022-28898-1

Figure Lengend Snippet: SARS-CoV-2 spike-specific antibody isotype and subclasses measured post-infection: a IgM, b IgA, c IgG1 and d IgG3. Antibody function measure post-SARS-CoV-2 infection: e antibody-dependent NK cell activation (ADNKA), f antibody-dependent neutrophil phagocytosis (ADNP), g antibody-dependent monocyte phagocytosis (ADMP) and h antibody-dependent complement deposition (ADCD). i Polar plot of various antibody isotype, subclass and function data, minimum-maximum normalised. The boxplots all display the median values with the first and third quartile, and the whiskers represent the highest and lowest values no more than 1.5 times the interquartile range from the corresponding hinge. A two-tailed Wilcoxon rank-sum test was used to compare between study time points. A generalised additive mixed model (GAMM) by restricted maximum likelihood—right-hand plots—was used to fit the immunological measures (log10 transformed) taken at multiple study time points, using Gaussian process smooth term. The GAMM plots the ribbon represents the 95% confidence interval around the fitted value. Disease severity group was included in the GAMM as a linear predictor and a participant identifier was included as a random effect. See Table for number of individuals evaluated per assay.

Article Snippet: For detection of anti-spike IgG2 and IgG4 steps modified as follows: (1) Plates were additionally coated with commercially available human immunoglobulin control (recombinant human IgG2 lambda or recombinant human IgG4 lambda (Bio-Rad)) to serve as internal controls, (2) Mouse anti-human IgG2 Fd-AP or mouse anti-human IgG4 Fc-AP (Southern Biotech) were used, and (3) Optical density at 405 nm was measured using an ELx808 absorbance reader (BioTek) until the immunoglobulin control reached a specified OD405.

Techniques: Infection, Activation Assay, Two Tailed Test, Transformation Assay