Journal: Journal of Biological Chemistry

Article Title: High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR

doi: 10.1074/jbc.m009483200

Figure Lengend Snippet: FIG. 1. Binding of anti-IgE E27 IgG1 to human FcgR. A, binding of E27 monomers (solid circles), dimeric (solid squares), and hexameric (open squares) complexes to FcgRIIA-Arg131. B, binding of E27 mono- mers (solid circles), dimeric (solid squares), and hexameric (open squares) complexes to FcgRIIIA-Phe158. Dimers were formed by mixing E27 IgG1 and a F(ab9)2 fragment of goat anti-human k light chain at 1:0.5 molar ratio at 25 °C for 1 h (50). Hexameric complexes (i.e. trim- eric in E27 IgG1) were formed by mixing E27 IgG1 with human IgE in a 1:1 molar ratio at 25 °C for 1 h (36).

Article Snippet: Plates were blocked with assay buffer at 25 °C for 1 h. Serial 3-fold dilutions of monomeric E27 and variants (10.0–0.0045 mg/ml) were added to plates and incubated for 2 h. After washing plates with assay buffer, IgG bound to FcgRI was detected with peroxidase-conjugated F(ab9)2 fragment of goat anti-human F(ab9)2-specific IgG (Jackson ImmunoResearch) or with peroxidase-conjugated protein G (Bio-Rad).

Techniques: Binding Assay

Journal: Cellular Logistics

Article Title: Intermolecular interactions involving an acidic patch on immunoglobulin variable domain and the γ2 constant region mediate crystalline inclusion body formation in the endoplasmic reticulum

doi: 10.1080/21592799.2017.1361499

Figure Lengend Snippet: Generation of a model human IgG mAb-3 possessing an acidic patch on the variable domain surface. (A) Schematic depiction of model mAbs. Two antibodies shown at the top are mAb-1 (IgG2κ) colored in orange and mAb-2 (IgG2λ) in green. Red circles located on the mAb-1's HC and mAb-2's LC represent the areas where negatively charged amino acids are densely located. Five Asp residues are in mAb-1 CDR-H3. Three Asp residues are in mAb-2 CDR-L2. The mAb-1 γ2-HC was paired with the mAb-2 λ-LC to generate mAb-3. Similarly, the mAb-1 κ-LC was paired with the mAb-2 γ2-HC to generate mAb-4 (shown in box). (B) A homology model showing the variable fragment of mAb-3. The surface is color-coded by the underlying amino acid residue charge in which red is negative, blue is positive, and white is neutral. VH, heavy chain variable domain. VL, light chain variable domain. (C) An overlay of the surface model and a corresponding ribbon diagram. Individual Asp residues comprising the prominent acidic patch are shown with the respective residue using the AHo numbering system33. Asp residues on CDR-H3 are shown in yellow background, while Asp residues on CDR-L2 are shown in green background.

Article Snippet: Affinity purified rabbit polyclonal anti-human IgG (H + L) polysera (cat. 309-005-082) used for Western blotting and Alexa Fluor® 594-conjugatged Fab fragment goat anti-human IgG (H + L) (cat. 109-587-003) used for imaging were from Jackson ImmunoResearch Laboratories.

Techniques:

Journal: Cellular Logistics

Article Title: Intermolecular interactions involving an acidic patch on immunoglobulin variable domain and the γ2 constant region mediate crystalline inclusion body formation in the endoplasmic reticulum

doi: 10.1080/21592799.2017.1361499

Figure Lengend Snippet: Model mAb-3 is secretion competent. (A) Cell culture media and whole cell lysate samples were prepared on day-7 post transfection and were subjected to SDS-PAGE followed by Western blot using rabbit anti-human IgG (H + L) polysera. Harvested cell culture medium was loaded and analyzed under reducing conditions (lanes 1–3) or non-reducing conditions (lanes 7–9). Whole cell lysates were loaded under reducing conditions (lanes 4–6). Expected band for heavy chain (HC), light chain (LC), or the whole IgG is marked by arrowhead and labeled accordingly. Anti-GAPDH blot is shown at the bottom as a loading reference. Both γ2-HC (lanes 2, 8) and λ-LC (lanes 3, 9) were secretion incompetent by themselves. The used constructs are shown at the top of corresponding lanes. (B) On day-2 post transfection, cell culture media were replaced with fresh growth media with or without 15 μg/ml BFA and maintained in suspension format for 24 hr until day-3 when the cell culture media and cell pellets were harvested and analyzed. The amount of mAb-3 protein secreted to the culture medium during the 24 hr BFA treatment is in lanes 1–2 (reducing conditions) and lanes 5–6 (non-reducing conditions). The amount of IgGs detected in the cell lysates is shown in lanes 3–4 (reducing conditions). Anti-GAPDH blot is shown as a loading reference for cell lysate samples. (C) Expression analysis of mAb-4. Cell culture media and whole cell lysate samples were prepared on day-7 post transfection and were analyzed as above. Expected band for HC, LC, or whole IgG is marked by arrowhead and labeled. (D) Expression comparison of mAb-3 and mAb-4. Cell culture media were harvested from mAb-3 and mAb-4 transfected cells on day-7 post transfection and analyzed under reducing (lanes 1, 2) or non-reducing (lanes 3, 4) conditions. SDS-PAGE gel was stained by Coomassie blue.

Article Snippet: Affinity purified rabbit polyclonal anti-human IgG (H + L) polysera (cat. 309-005-082) used for Western blotting and Alexa Fluor® 594-conjugatged Fab fragment goat anti-human IgG (H + L) (cat. 109-587-003) used for imaging were from Jackson ImmunoResearch Laboratories.

Techniques: Cell Culture, Transfection, SDS Page, Western Blot, Labeling, Construct, Expressing, Staining

Journal: Cellular Logistics

Article Title: Intermolecular interactions involving an acidic patch on immunoglobulin variable domain and the γ2 constant region mediate crystalline inclusion body formation in the endoplasmic reticulum

doi: 10.1080/21592799.2017.1361499

Figure Lengend Snippet: Needle-like crystalline inclusions co-localize with ER resident proteins. Fluorescent micrographs of HEK293 cells transfected to express mAb-3. On day-2 post transfection, HEK293 cells were resuspended in fresh cell culture media containing 15 μg/ml BFA, then immediately seeded onto poly-lysine coated glass coverslips and statically cultured for 24 hr. On day-3, cells were fixed, permeabilized, and immuno-stained. (A) Co-staining was performed by using Alexa Fluor 488-conjugated Protein A and Alexa Fluor 594-conjugated Fab fragments generated from goat anti-human IgG (H + L) polysera. (B, C) Transfected cells were co-stained with Alexa Fluor 488-conjugated Protein A and anti-ERp57 (B) or anti-BiP (C). Green and red image fields were superimposed to create ‘merge’ views. Scale bars represent 10 μm.

Article Snippet: Affinity purified rabbit polyclonal anti-human IgG (H + L) polysera (cat. 309-005-082) used for Western blotting and Alexa Fluor® 594-conjugatged Fab fragment goat anti-human IgG (H + L) (cat. 109-587-003) used for imaging were from Jackson ImmunoResearch Laboratories.

Techniques: Transfection, Cell Culture, Staining, Generated

Journal: Cellular Logistics

Article Title: Intermolecular interactions involving an acidic patch on immunoglobulin variable domain and the γ2 constant region mediate crystalline inclusion body formation in the endoplasmic reticulum

doi: 10.1080/21592799.2017.1361499

Figure Lengend Snippet: IgG1 version of mAb-3 does not induce crystalline inclusion bodies. (A) Cell culture media were harvested on day-7 post transfection and were subjected to SDS-PAGE under reducing conditions (lanes 1–5) or non-reducing conditions (lanes 11–15). Cell lysates were analyzed under reducing conditions (lanes 6–10). Western blotting was performed using anti-human IgG (H + L) polysera. Constructs used are shown at the top of corresponding lanes. Expected band for HC, LC, or the whole IgG is marked by arrowhead and labeled. (B) The same set of samples analyzed in panel A were stained by Coomassie blue dye. (C, D) Fluorescent micrographs of HEK293 cells expressing the IgG1 version of mAb-3. On day-2 post transfection, HEK293 cells were resuspended in fresh cell culture media with or without 15 μg/ml BFA, then seeded onto poly-lysine coated glass coverslips and statically cultured for 24 hr. On day-3, cells were fixed, permeabilized, and immuno-stained. Co-staining was performed by using FITC-conjugated anti-gamma chain and Texas Red-conjugated anti-lambda chain polyclonal antibodies. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to generate ‘overlay’ views. (E) Fluorescent micrographs of HEK293 cells transfected with the γ1 version of mAb-3 HC construct alone. On day-3, cells were fixed, permeabilized, and co-stained with anti-CD147 and anti-gamma chain. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘red’ were superimposed to generate ‘overlay’ views.

Article Snippet: Affinity purified rabbit polyclonal anti-human IgG (H + L) polysera (cat. 309-005-082) used for Western blotting and Alexa Fluor® 594-conjugatged Fab fragment goat anti-human IgG (H + L) (cat. 109-587-003) used for imaging were from Jackson ImmunoResearch Laboratories.

Techniques: Cell Culture, Transfection, SDS Page, Western Blot, Construct, Labeling, Staining, Expressing

Journal: Cellular Logistics

Article Title: Intermolecular interactions involving an acidic patch on immunoglobulin variable domain and the γ2 constant region mediate crystalline inclusion body formation in the endoplasmic reticulum

doi: 10.1080/21592799.2017.1361499

Figure Lengend Snippet: Stepwise neutralization of acidic surface patch progressively diminishes mAb-3 secretion. HEK293 cells were co-transfected with the parental subunits or the Asp-to-Ala mutated subunits in various combination. Cell culture media were harvested on day-7 post transfection and were analyzed by SDS-PAGE under reducing (A) or non-reducing (B) conditions followed by Coomassie blue staining. Whole cell lysates were loaded under reducing conditions (C) followed by Western blotting using anti-human IgG (H + L) polysera. Expected band for HC, LC, or the whole IgG is marked by arrowhead and labeled. Anti-GAPDH blot is shown as a loading reference for cell lysate samples.

Article Snippet: Affinity purified rabbit polyclonal anti-human IgG (H + L) polysera (cat. 309-005-082) used for Western blotting and Alexa Fluor® 594-conjugatged Fab fragment goat anti-human IgG (H + L) (cat. 109-587-003) used for imaging were from Jackson ImmunoResearch Laboratories.

Techniques: Neutralization, Transfection, Cell Culture, SDS Page, Staining, Western Blot, Labeling