Journal: Journal of Cellular and Molecular Medicine

Article Title: Elevation of IGF-2 receptor and the possible underlying implications in end-stage heart failure patients before and after heart transplantation

doi: 10.1111/j.1582-4934.2011.01414.x

Figure Lengend Snippet: Western blot analysis of cardiac IGF-2R. (A) Representative Western blot analysis of cardiac IGF-2R expression. (B) Densitometric quantification of cardiac IGF-2R expression. The IGF-2R protein levels related to the internal standard protein GAPDH were calculated as the relative abundance. Densitometric analyses of the blots showed an apparent increase in IGF-2R in DCM failing hearts (DCM-HF, n = 5) compared with non-failing control hearts (NF, n = 5; ** P < 0.01). Data are presented as mean ± S.D.

Article Snippet: Sections were then incubated with primary antibodies for 1 hr at room temperature and washed in PBS buffer for 10 min., followed by incubation with IgG-peroxidase conjugated secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) for 1 hr at room temperature, washed in PBS buffer for 10 min., and incubated with 0.5 mg/ml diaminobenzidine tetrahydrochloride 2-hydrate plus 0.05% H 2 O 2 for 5 min. Primary antibodies used included goat anti-human IGF-2R antibody (2 μg/ml, Cat. No. AF2447; R&D System Inc., MN, USA) and rabbit anti-human granzyme B polyclonal antibodies (1:150, Cat. No. Z2145; ZETA Corporation, CA, USA).

Techniques: Western Blot, Expressing, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Elevation of IGF-2 receptor and the possible underlying implications in end-stage heart failure patients before and after heart transplantation

doi: 10.1111/j.1582-4934.2011.01414.x

Figure Lengend Snippet: Histopathological analysis of IGF-2R. (A) Representative light microscopic findings in haematoxylin and eosin staining. The normal appearance of myocardial fibres with central nuclei is seen in non-failing control hearts (NF) and interstitial fibrosis replacement is found in DCM failing hearts (DCM-HF). (B) Representative light microscopic IGF-2R immunoreactivity using a monoclonal antibody, which recognizes IGF-2R. Few and weak immunoreactivity of IGF-2R is observed in non-failing control hearts (NF), but more and strong IGF-2R immunoreactivity can be seen in DCM failing hearts (DCM-HF). Arrows indicated the positive staining.

Article Snippet: Sections were then incubated with primary antibodies for 1 hr at room temperature and washed in PBS buffer for 10 min., followed by incubation with IgG-peroxidase conjugated secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) for 1 hr at room temperature, washed in PBS buffer for 10 min., and incubated with 0.5 mg/ml diaminobenzidine tetrahydrochloride 2-hydrate plus 0.05% H 2 O 2 for 5 min. Primary antibodies used included goat anti-human IGF-2R antibody (2 μg/ml, Cat. No. AF2447; R&D System Inc., MN, USA) and rabbit anti-human granzyme B polyclonal antibodies (1:150, Cat. No. Z2145; ZETA Corporation, CA, USA).

Techniques: Staining, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Elevation of IGF-2 receptor and the possible underlying implications in end-stage heart failure patients before and after heart transplantation

doi: 10.1111/j.1582-4934.2011.01414.x

Figure Lengend Snippet: ELISA of serum IGF-2R and serum CD8. (A) The mean level of serum IGF-2R in DCM heart failure patients before heart transplantation (Bef-Htx, n = 11) was significantly higher than that in non-failing control subjects (NF, n = 11). After heart transplantation, serum IGF-2R levels further increased, peaked at the first month, and gradually reduced close to the pre-operative level at the 6th months, but remained to be higher than that in non-failing controls. ** P < 0.01 versus NF. ## P < 0.01 versus Bef-Htx. (B) The mean level of serum CD8 in DCM heart failure patients before heart transplantation (Bef-Htx, n = 11) had no increase when compared with that in non-failing control subjects (NF, n = 11). Mean levels of serum CD8 also had no change at different time point after heart transplantation compared with that before heart transplantation (Bef-Htx). Data are presented as mean ± S.D.

Article Snippet: Sections were then incubated with primary antibodies for 1 hr at room temperature and washed in PBS buffer for 10 min., followed by incubation with IgG-peroxidase conjugated secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) for 1 hr at room temperature, washed in PBS buffer for 10 min., and incubated with 0.5 mg/ml diaminobenzidine tetrahydrochloride 2-hydrate plus 0.05% H 2 O 2 for 5 min. Primary antibodies used included goat anti-human IGF-2R antibody (2 μg/ml, Cat. No. AF2447; R&D System Inc., MN, USA) and rabbit anti-human granzyme B polyclonal antibodies (1:150, Cat. No. Z2145; ZETA Corporation, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Transplantation Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Elevation of IGF-2 receptor and the possible underlying implications in end-stage heart failure patients before and after heart transplantation

doi: 10.1111/j.1582-4934.2011.01414.x

Figure Lengend Snippet: Immunohistochemical detection of IGF-2R and granzyme B-positive T lymphocytes. Representative immunohistochemical staining on the serial sections from biopsies with rejection grade 3 from patients #6 (A) and from biopsies with rejection grade 2 from patient #4 (B) at the 3 rd month after heart transplantation showed coexistence of IGF-2R and granzyme B-positive T lymphocytes, accompanied by mononuclear cell infiltration with myocyte damage in the same sites in haematoxylin and eosin (HE) staining.

Article Snippet: Sections were then incubated with primary antibodies for 1 hr at room temperature and washed in PBS buffer for 10 min., followed by incubation with IgG-peroxidase conjugated secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) for 1 hr at room temperature, washed in PBS buffer for 10 min., and incubated with 0.5 mg/ml diaminobenzidine tetrahydrochloride 2-hydrate plus 0.05% H 2 O 2 for 5 min. Primary antibodies used included goat anti-human IGF-2R antibody (2 μg/ml, Cat. No. AF2447; R&D System Inc., MN, USA) and rabbit anti-human granzyme B polyclonal antibodies (1:150, Cat. No. Z2145; ZETA Corporation, CA, USA).

Techniques: Immunohistochemical staining, Staining, Transplantation Assay

Journal: Molecular Brain

Article Title: Insulin and IGF1 signalling pathways in human astrocytes in vitro and in vivo ; characterisation, subcellular localisation and modulation of the receptors

doi: 10.1186/s13041-015-0138-6

Figure Lengend Snippet: Schematic of the insulin/IGF1R signalling pathway. A simplified representation of the insulin/IGF1 signalling pathway depicting the downstream activation of Akt and p44-42 MAPK through binding of insulin or IGF1 to their respective receptors. Furthermore insulin can bind to IGF1R and IGF1 to IR and IR α-subunits/ β-subunits can form heterodimers with IGF1R α-subunits /β-subunits adding further complexity at the level of the receptors. There are also numerous downstream feedback loops within this and other signalling pathways which act to regulate signalling through this pathway at multiple levels

Article Snippet: To impair signalling through IGF1R cultures were treated with 11 μg/ml IGF1 receptor monoclonal antibody (24 h) (MAB391, R&D Systems, MN, USA).

Techniques: Activation Assay, Binding Assay

Journal: Molecular Brain

Article Title: Insulin and IGF1 signalling pathways in human astrocytes in vitro and in vivo ; characterisation, subcellular localisation and modulation of the receptors

doi: 10.1186/s13041-015-0138-6

Figure Lengend Snippet: Insulin/IGF1 pathway characterisation in astrocytes. Immunoblots of astrocytes cultured in either serum containing or serum deprived media with additional supplementation from either a recombinant human 1 μM insulin or b 11.2nM recombinant human IGF1. Respresentative images from blots probed with antibodies against IRβ, IGF1Rβ, IRS1, IRS2, pAkt, Total Akt, p44/42 MAPK are shown. *α-tubulin was used as a loading control for blots and a representative loading control is shown. Molecular weight markers are indicated (kDa)

Article Snippet: To impair signalling through IGF1R cultures were treated with 11 μg/ml IGF1 receptor monoclonal antibody (24 h) (MAB391, R&D Systems, MN, USA).

Techniques: Western Blot, Cell Culture, Recombinant, Control, Molecular Weight

Journal: Molecular Brain

Article Title: Insulin and IGF1 signalling pathways in human astrocytes in vitro and in vivo ; characterisation, subcellular localisation and modulation of the receptors

doi: 10.1186/s13041-015-0138-6

Figure Lengend Snippet: Impairment of IGF1 signalling using a monoclonal IGF1 antibody (MAB391). Human astrocytes treated with 11 μg/ml MAB391 for 24 h ( a ). Representative immunoblots demonstrate reductions in IGF1Rβ, IRβ and pAkt in response to MAB391 with no impact on total IRS1 or downstream signalling through p44/42 MAPK. *α-tubulin was used as a loading control for blots and a representative loading control is shown. Molecular weight markers are indicated (kDa). b Bar charts show quantification of immunoblots, pAkt was normalised to Akt/α-tubulin. c Immunofluorescence showing the reduction in IGF1R, scale bars represent 10 μM. d qPCR analysis of IGF1Rβ RNA after 24 h show no differences at the RNA level. Data represents mean + SEM ( n = 3, 3 replicates/experiments, Unpaired t -test, ** p < 0.01)

Article Snippet: To impair signalling through IGF1R cultures were treated with 11 μg/ml IGF1 receptor monoclonal antibody (24 h) (MAB391, R&D Systems, MN, USA).

Techniques: Western Blot, Control, Molecular Weight, Immunofluorescence

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Cartoon representation, SDS-PAGE, reduced western blot and SEC-MALS analyses of the iMab-EI. (A) Cartoon representation of the iMab-EI with linkers connecting the antibody domains shown as red dotted lines. (B) Reduced SDS-PAGE (lanes 1 to 4) and reduced western blot analysis (lanes 5 to 16) of iMab-EI, anti-EGFR and anti-IGF1R antibodies. Molecular mass standards are schematically shown. Lκ, Lλ, H1 and H2 denote anti-EGFR kappa light chain, anti-IGF1R lambda light chain, anti-EGFR heavy chain, anti-IGF1R heavy chain, respectively. FL denotes the full-length iMab-EI. Lane 2 is the anti-EGFR antibody, lane 3 is the anti-IGF1R antibody and lane 4 is the full-length iMab-EI. Lanes 6, 7 and 8 is the reduced western blot probed with an anti-human Fc antibody. Lanes 10, 11 and 12 is the reduced western blot probed with an anti-human kappa antibody. Lane 14, 15 and 16 is the reduced western blot probed with an anti-human lambda antibody. (C) SEC-MALS of the iMab-EI after protein A purification. (D) SEC-MALS of monomeric iMab-EI. The molecular weights in KDa were obtained using SEC-MALS.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: SDS Page, Western Blot, Purification

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Reduced SDS-PAGE, reduced western-blot, SEC-MALS, rRP-HPLC and rLCMS of the iMab-EI after thrombin cleavage. (A) Reduced SDS-PAGE (lanes 1 to 4) and western blot analysis (lanes 5 to 16) of iMab-EI, anti-EGFR and anti-IGF1R antibodies. Molecular mass standards are schematically shown. Lκ, Lλ, H1 and H2 denote anti-EGFR kappa light chain, anti-IGF1R lambda light chain, anti-EGFR heavy chain, anti-IGF1R heavy chain, respectively. Lκ*, Lλ*, H1* and H2* denote the iMab-EI anti-EGFR kappa light chain, anti-IGF1R lambda light chain, anti-EGFR heavy chain, and anti-IGF1R heavy chain, respectively. Lane 2 is the anti-EGFR antibody, lane 3 is the anti-IGF1R antibody and lane 4 is the iMab-EI. Lanes 6, 7 and 8 is the reduced western blot probed with an anti-human Fc antibody. Lanes 10, 11 and 12 is the reduced western blot probed with an anti-human kappa antibody. Lanes 14, 15 and 16 is the reduced western blot probed with an anti-human lambda antibody. The chains of the iMab-EI are migrating slower compared to the chains from the 2 parental antibodies due their slightly higher molecular weight. (B) SEC-MALS analysis of the iMab-EI after thrombin cleavage showed that the iMab-EI is 99% monomer and has a molecular weight resembling that of an intact IgG1. (C) rRP-HPLC of the iMab-EI, (D) anti-IGF1R, and (E) anti-EGFR. The identity of each peak is schematically labeled as shown in panel A. (F) rLCMS of iMab-EI. The chains of the iMab-EI are labeled as shown in the panel A and as in panel C. The molecular masses of the 4 chains are shown in Daltons. The rLCMS was performed after Endo S treatment to trim the N-glycan found at the conserved N297 site in iMab-EI CH2 domain.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: SDS Page, Western Blot, Molecular Weight, Labeling

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: iMab-EI has native interchain disulfide bonds at the hinge and at the heavy-light chains as demonstrated using non-reduced SDS-PAGE. Lane 1 is the molecular weight standards; lane 2 is the anti-IGF1R, lane 3 is the anti-EGFR.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: SDS Page, Molecular Weight

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Non-reduced LCMS of the iMab-EI and the 2 parental antibodies intact glycosylated and after treatment with IdeS, which release the Fc fragment from the F(ab)’2. (A) Intact glycosylated iMab-EI; (B) IdeS-treated iMab-EI; (C) Intact glycosylated anti-EGFR; (D) IdeS-treated anti-EGFR; (E) Intact glycosylated anti-IGF1R; (F) IdeS-treated anti-IGF1R; The deconvoluted mass in Dalton is shown on the x-axis and the ions counts are shown on the y-axis. The molecular weight of each peak is schematically shown with the corresponding glycoform. LCMS show the iMab-EI has correctly formed the interchain disulfide bonds at the hinge and at the heavy and light chains similar to IgG1 parental antibodies.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Molecular Weight

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Isoelectric point (pI) of the iMab-EI and the 2 parental antibodies. (A) iMab-EI pI main peak is 8.43; (B) anti-EGFR antibody pI main peak is 7.72; (C) anti-IGF1R pI main peak is 8.24. pI marker peaks 4.22 and 9.77 are schematically shown.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Marker

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Binding of iMab-EI to EGFR and IGF1R using BIAcore. (A) Concurrent binding to antigens using BIAcore. iMab-EI was immobilized on the BIAcore sensor chip and dual binding was determined by first injecting IGF1R, followed by co-injection of IGF1R and EGFR. (B) Binding kinetics to IGF1R of the iMab-EI and the Fab prepared from the anti-IGF1R antibody. (C) Binding kinetics to EGFR of the iMab-EI and the Fab prepared from the anti-EGFR antibody.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Binding Assay, Injection

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Differential scanning calorimetry (DCS) of the iMab-EI (A), the anti-EGFR (B) and anti-IGF1R (C) antibodies. Transition temperatures are shown as Tm in°C. DSC analysis shows that the iMab-EI has transition temperatures similar to the transition temperatures of the anti-EGFR and anti-IGF1R antibodies, indicating the iMab-EI has a structure similar to the anti-EGFR and anti-IGF1R antibodies. (D) Hydrodynamic radios (Rh) determined using SEC-MALS of the iMab-EI at 0.5, 2 and 6 mg/mL. The Rh of the iMab-EI remains similar at the 3 concentrations tested, which is an indication of structural stability of the iMab-EI.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Differential Scanning Calorimetry

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Schematic representation and data of the EGFR ELISA transfer assay. (A) EGFR was immobilized on the ELISA plate, (B) and incubated with iMab-EI or the 2 parental antibodies. The well volume of the iMab-EI or the parental antibodies pre-absorbed on an EGFR-coated ELISA plates (C) were transferred to an ELISA plate with immobilized IGF1R, (D) followed by detection of binding using an anti-human-lambda-HRP labeled antibody.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Labeling

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: Schematic representation and data of the IGF1R ELISA transfer assay. (A) IGF1R was immobilized on the ELISA plate, (B) and incubated with iMab-EI or the 2 parental antibodies. The well volume of the iMab-EI or the parental antibodies pre-absorbed on an IGF1R-coated ELISA plates (C) were transferred to an ELISA plate with immobilized EGFR, (D) followed by detection of binding using an anti-human-kappa-HRP labeled antibody.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Labeling

Journal: mAbs

Article Title: Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

doi: 10.1080/19420862.2016.1277301

Figure Lengend Snippet: In vivo efficacy of iMab-EI using a xenograft mouse model of a patient derived non-small cell lung cancer. iMab-EI (red curve), isotype non-binding antibody (black curve) and combination of the anti-EGFR and anti-IGF1R antibodies (green curve) were dosed at 10 mg/kg 2 times weekly for 2 weeks. Vehicle-treated mice (blue curve) were used as negative control.

Article Snippet: Two µg/mL (30 µL/well) of EGFR (R&D system, cat. No. 236-EG-200) or IGF1R (R&D System, cat. No. 391-GR-050) prepared in PBS, pH 7.2, were coated on ELISA plates overnight at 4°C.

Techniques: In Vivo, Derivative Assay, Binding Assay, Negative Control