Human Anti-ERBB2 Recombinant Antibody (clone bH1) (CAT#: PABZ-041)

Figure 1 Analysis of BH-antibody affinities for recombinant ERBB2 by ELISA.

50 ng/well ERBB2 ECD-coated Nunc-polysorb plates were incubated with increasing concentrations of purified anti-ERBB2 antibodies. Strength of antibody binding was colorimetrically detected (OD 405 nm) by the alkaline phosphatase activity following the incubation with alkaline phosphatase-conjugated anti-mouse IgG antibodies. T Bars: SD.

Ceran, C. (2012). Novel monoclonal antibodies targeting conformational ERBB2 epitopes (Doctoral dissertation, Bilkent University).

Figure 2 Analysis of BH-antibody affinities for cellular ERBB2 by Flow Cytometry (B).

~3x10⁵ T47D (left column) or ~5x10⁵ BT-474 (right column) cells were incubated with increasing amount of BH-antibodies (0.5 – 2 μg antibody/sample) in 100 μl. Anti-ERBB2 incubation of paraformaldehyde fixed single cell suspensions was followed by Alexa-488-conjugated secondary antibodies and fluorescence intensities were measured using flow cytometry. Each graph shows fluorescence intensities of cells stained with different primary antibodies used at same concentration. Maximum fluorescence emissions at 488 nm are shown on x-axis (FL1-H) and cell counts are shown on y-axis. IgG1-isotype control antibody (IgG) at equal concentrations were used as negative controls. (-) indicates the control samples which were incubated only with Alexa-488-conjugated secondary antibodies without any prior primary antibody incubation.

Ceran, C. (2012). Novel monoclonal antibodies targeting conformational ERBB2 epitopes (Doctoral dissertation, Bilkent University).

Figure 3 Analysis of cellular ERBB2 by indirect immunofluorescence using BH-antibodies.

A) For FISH analysis, human breast cancer cells with different ERBB2 levels were harvested at confluence, fixed and subjected to dual-color FISH using Texas Red-labeled ERBB2 (red) and FITC-labeled chromosome 17 α-satellite CEP17 (green) DNA probes. B-G) For immunofluorescence assays, cells were seeded on coverslips on 6-well plates and incubated 36 hours in cell culture medium at 37°C. Paraformaldehyde fixed and permeabilised cells were incubated with 20 μg/ml BH-antibodies for two hours. Photographs are representative of merged images of BH-antibody binding detected using Alexa-488-conjugated anti-mouse IgG and nuclei counterstained with DAPI. (-) indicates the control samples where the cells were incubated only with Alexa-488-conjugated secondary antibodies without any prior primary antibody incubation.

Ceran, C. (2012). Novel monoclonal antibodies targeting conformational ERBB2 epitopes (Doctoral dissertation, Bilkent University).

Figure 4 Immunoprecipitation of endogenous ERBB2 by BH-antibodies.

Endogenously expressed ERBB2 present in 2 mg T47D RIPA lysate was immunoprecipitated by 20 μg BH-antibodies or Trastuzumab (Tzm). Antigen-antibody complexes that were captured onto Protein G-conjugated beads were eluted by boiling in SDS-PAGE loading dye (left panel). ERBB2 proteins that were uncaptured by the antibody-bead complex remained in the flow through (right panel). Proteins resolved under denaturing conditions were subjected to western blot assay using CB11 antibody. Trastuzumab was used as a positive control for ERBB2 immunoprecipitation. (-) is the negative control where T47D lysate was incubated with Protein-G beads in the absence of any antibody. ERBB2 protein band is detected at 185 kDa.

Ceran, C. (2012). Novel monoclonal antibodies targeting conformational ERBB2 epitopes (Doctoral dissertation, Bilkent University).

Figure 5 Analysis of senescence induction upon BH1 treatment in the presence or absence of TNF-α.

SK-BR-3 cells plated on coverslips in 12-well dishes were treated with 5 μg/ml BH1, Trastuzumab or IgG1 isotype control antibody with or without 1000 U/ml TNF-α. After 6 day treatment, cells were formaldehyde fixed and SAβ-gal stained for 12 hours. Nuclei was counterstained with nuclear fast red and and cells were observed under light microscopy. SAβ-gal positive cells contain dark blue spots suggesting a senescent phenotype. 100 μM CPT (camptothecin) and 50 ng/ml Adriamycin were used as positive controls of senescence induction.

Ceran, C. (2012). Novel monoclonal antibodies targeting conformational ERBB2 epitopes (Doctoral dissertation, Bilkent University).