Journal: Allergy

Article Title: Allergic Reactivity and Memory Occur Independently of Sequential Switching Through IgG1.

doi: 10.1111/all.16460

Figure Lengend Snippet: FIGURE 5 | Memory B cells retain type 2 polarization in the absence of IgG1 expression. (A–L) IgG1-def and wild-type (WT) mice were sensitized to ovalbumin (OVA) and memory B cell (MBC) number and phenotype were assessed 1 month later. Spleen and mesenteric lymph node samples were pooled prior to OVA-tetramer enrichment. (A and B) Representative flow cytometry plots and number of OVA-specific isotype switched MBCs. (C–H) Representative flow cytometry plots depicting IgG subtype expression (C and F) and plots summarizing the frequency (left) and number (right) of IgG MBCs (D and E, G and H). Pre-gate: B220+ CD3− F4/80− OVA Tetramer+ Control Tetramer− GL7− CD38+ IgM− IgD−. (I and J) Representative flow cytometry plots of CD23 and IL-4Rα expression (I) and summary plots (J) depicting the frequency and number of MBC2s. Pre-gate: B220+ CD3− F4/80− OVA Tetramer+ Control Tetramer− GL7− CD38+ IgM− IgD−. (K and L) Representative flow cytometry plots (K) and summary plot of frequency (L) of IgG3 expression among MBC2s. Pre-gate: B220+ CD3− F4/80− OVA Tetramer+ Control Tetramer− GL7− CD38+ IgM− IgD− CD23hi IL-4Rαhi. **p < 0.01; ***p < 0.001; ****p < 0.0001 (unpaired t test). Dots represent samples from individual mice and bars within groups represent the mean. Data are pooled from two independent experiments.

Article Snippet: The next day, plates were washed 3 times and incubated with 50 μL of either 0.25 μg/mL biotinylated anti- mouse IgG1 (Southern Biotech, 1070–08) or biotinylated anti- mouse IgG (H + L) (Poly4053, Biolegend, 405,301) for 2 h at RT.

Techniques: Expressing, Flow Cytometry, Control

Journal: RSC Advances

Article Title: The effect of Fc region affinity of protein-based antibody-recruiting molecules on antibody-dependent cellular cytotoxicity †

doi: 10.1039/d4ra03391d

Figure Lengend Snippet: (A) Structure of Fc-ARM containing a target-binding terminus (TBT) and antibody-binding terminus (ABT). (B) Schematic illustration showing the activation of an NK cell by Fc-ARM via the crosslinking of FcγRIIIa and antigen. A complex model of HER2/Fc-ARM/IgG/FcγRIIIa is shown in Fig. S3. (C) 3D structure predictions of two types of Fc-ARMs. Z HER2:342 and Z domain were used as the TBT (red) and ABT (orange), respectively.

Article Snippet: Affinity measurements were performed against biotinylated human IgG1 Fc protein (IG1-H8213, ACROBiosystems), biotinylated human HER2/ErbB2 protein (HE2-H82 × 10 2 , ACROBiosystems), and biotinylated human EGFR protein (EGR-H82 × 10 3 , ACROBiosystems) immobilized on Octet® SA Biosensor (18-5019, Sartorius), using an Octet RED96 System (ForteBio), following the manufacturer's instructions.

Techniques: Binding Assay, Activation Assay

Journal: RSC Advances

Article Title: The effect of Fc region affinity of protein-based antibody-recruiting molecules on antibody-dependent cellular cytotoxicity †

doi: 10.1039/d4ra03391d

Figure Lengend Snippet: Recruitment of fluorescence-labeled IgG (IgG-Fluor) via A-Z or A-ZZ to SK-BR-3 and MDA-MB-231 cells, which are HER2 positive and negative, respectively. Cells were seeded and incubated overnight. Fluorescence-labeled IgG (1.0 μM) and each Fc-ARM (100 nM) were added. Nuclei were stained with Hoechst 33 342 (blue). Scale bar = 20 μm.

Article Snippet: Affinity measurements were performed against biotinylated human IgG1 Fc protein (IG1-H8213, ACROBiosystems), biotinylated human HER2/ErbB2 protein (HE2-H82 × 10 2 , ACROBiosystems), and biotinylated human EGFR protein (EGR-H82 × 10 3 , ACROBiosystems) immobilized on Octet® SA Biosensor (18-5019, Sartorius), using an Octet RED96 System (ForteBio), following the manufacturer's instructions.

Techniques: Fluorescence, Labeling, Incubation, Staining

Journal: RSC Advances

Article Title: The effect of Fc region affinity of protein-based antibody-recruiting molecules on antibody-dependent cellular cytotoxicity †

doi: 10.1039/d4ra03391d

Figure Lengend Snippet: The effect of the concentration of Fc-ARMs and fluorescence-labeled IgG on ternary complex formation on SK-BR-3. (A) and (B) SK-BR-3 cells were treated with increasing concentrations of Fc-ARM with an excess amount of fluorescence-labeled IgG (1.0 μM). (C) and (D) SK-BR-3 cells were treated with increasing concentrations of IgG with Fc-ARM at a concentration of 10 nM. The y -axis indicates the median fluorescence intensity and the x -axis indicates the concentration of each sample. Two experimental repeats were performed.

Article Snippet: Affinity measurements were performed against biotinylated human IgG1 Fc protein (IG1-H8213, ACROBiosystems), biotinylated human HER2/ErbB2 protein (HE2-H82 × 10 2 , ACROBiosystems), and biotinylated human EGFR protein (EGR-H82 × 10 3 , ACROBiosystems) immobilized on Octet® SA Biosensor (18-5019, Sartorius), using an Octet RED96 System (ForteBio), following the manufacturer's instructions.

Techniques: Concentration Assay, Fluorescence, Labeling

Journal: RSC Advances

Article Title: The effect of Fc region affinity of protein-based antibody-recruiting molecules on antibody-dependent cellular cytotoxicity †

doi: 10.1039/d4ra03391d

Figure Lengend Snippet: ADCC induced by the combination of Fc-ARM and non-target IgG. SK-BR-3 cells were treated with Fc-ARM (A) A-Z and (B) A-ZZ, and IgG. Fc-ARM (10 nM) was added to the cells and incubated for 30 min. After unbound molecules were washed away, IgG (100 nM) was added. The y -axis indicates the cellular cytotoxicity ratio and the x -axis indicates the ratio of the number of NK cells to cancer cells.

Article Snippet: Affinity measurements were performed against biotinylated human IgG1 Fc protein (IG1-H8213, ACROBiosystems), biotinylated human HER2/ErbB2 protein (HE2-H82 × 10 2 , ACROBiosystems), and biotinylated human EGFR protein (EGR-H82 × 10 3 , ACROBiosystems) immobilized on Octet® SA Biosensor (18-5019, Sartorius), using an Octet RED96 System (ForteBio), following the manufacturer's instructions.

Techniques: Incubation

Journal: Clinical & Translational Immunology

Article Title: Immunoglobulin G genetic variation can confound assessment of antibody levels via altered binding to detection reagents

doi: 10.1002/cti2.1494

Figure Lengend Snippet: (a) Structure of an IgG1 antibody. Immunoglobulins comprise two fragment antigen‐binding (Fab) regions and a fragment crystallisable (Fc) region connected by a flexible hinge. Two constant heavy (C H ) and two constant light (C L ) chains are connected via disulphide bridges, which, along with two variable heavy (V H ) and two variable light (V L ) chains, form an ~150 kDa molecule. The C H chain comprises three structural regions (C H 1‐C H 3) as well as the flexible hinge. (b) IgG polymorphisms giving rise to allotypes are located within the C H 1 and C H 3 regions of IgG1. EC 50 s of (c) 4E3, (d) HP6001, (e) HP6069 and (f) MTG1218 anti‐IgG1 clones for kappa and lambda variants of G1m‐1,3 and G1m1,17 allotype mAbs. Measurements were performed in triplicate and mean values ± SEM are indicated. Curves were fitted using a four‐parameter nonlinear regression. Mann–Whitney U ‐tests were performed between responses against G1m1,17 (kappa and lambda) and G1m‐1,3 (kappa and lambda) allotype mAb standards within each concentration of 4E3 anti‐IgG1 detection reagent. P < 0.01 (**); P < 0.05 (*).

Article Snippet: PE‐conjugated mouse anti‐human IgG1 (4E3 (Southern Biotech, 9052‐09) or HP6001 (Southern Biotech, 9054‐09)), PE‐conjugated mouse anti‐human pan ‐IgG (JDC‐10 (Southern Biotech, 9040‐09)), or biotinylated mouse anti‐human IgG1 (HP6069 (Merck, 411543‐200UG) or MTG1218 (MabTech, 3851‐14‐250)) were diluted to 1.3 μg mL −1 and added at 25 μL per well before incubation for 2 h at room temperature on a plate shaker.

Techniques: Binding Assay, Clone Assay, MANN-WHITNEY, Concentration Assay

Journal: Clinical & Translational Immunology

Article Title: Immunoglobulin G genetic variation can confound assessment of antibody levels via altered binding to detection reagents

doi: 10.1002/cti2.1494

Figure Lengend Snippet: Plasma IgG1 levels of two‐dose BNT162b2 vaccinees against (a) SARS‐CoV‐2 S Trimer, (b) SARS‐CoV‐2 S2, (c) SARS‐CoV‐2 S1, (d) SARS‐CoV‐2 RBD and (e) H1 (Cal/09) measured with 4E3, HP6001, HP6069, or MTG1218 anti‐IgG1 clones. Mann–Whitney U ‐tests were performed between G1m1,17/G1m1,17 and G1m‐1,3/G1m‐1,3 individuals within each anti‐IgG1 clone. Fold changes indicate relative increases in antigen‐specific IgG1 detected in G1m1,17/G1m1,7 compared to G1m‐1,3/G1m‐1,3 vaccinees. Correlations between anti‐SARS‐CoV‐2 IgG1 levels measured with each of 4E3, HP6001, HP6069 and MTG1218 anti‐IgG1 clones for representative SARS‐CoV‐2 antigens (f) S Trimer, (g) S1. The Shapiro–Wilk test confirmed normally of data distribution and the Pearson correlation coefficient ( r ) was computed. Black dots represent G1m‐1,3/G1m‐1,3 individuals ( n = 11); Blue dots represent G1m1,17/G1m1,17 individuals ( n = 7). P < 0.0001 (****); non‐significant (ns).

Article Snippet: PE‐conjugated mouse anti‐human IgG1 (4E3 (Southern Biotech, 9052‐09) or HP6001 (Southern Biotech, 9054‐09)), PE‐conjugated mouse anti‐human pan ‐IgG (JDC‐10 (Southern Biotech, 9040‐09)), or biotinylated mouse anti‐human IgG1 (HP6069 (Merck, 411543‐200UG) or MTG1218 (MabTech, 3851‐14‐250)) were diluted to 1.3 μg mL −1 and added at 25 μL per well before incubation for 2 h at room temperature on a plate shaker.

Techniques: Clone Assay, MANN-WHITNEY