goat anti mouse adam8 ab  (R&D Systems)

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: ADPs bind to native ADAM8. Flow cytometry was performed using HEK293 cells expressing full-length ADAM8 (HEK-A8) vs. an empty vector control DNA (HEK-EV) and decreasing antibody (Ab) concentrations. Mean Fluorescent Intensity (MFI) indicates extent of binding of each Ab.

Article Snippet: As negative and positive controls, 1 μg/mL normal mouse IgG and goat anti-mouse ADAM8 Ab (AF1031, R&D Systems, RRID: AB_354549), respectively, were analyzed.

Techniques: Flow Cytometry, Expressing, Plasmid Preparation, Control, Binding Assay

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: ADPs bind with high affinity to recombinant human ADAM8 (rHuADAM8). The half-maximal effective concentration (EC50) obtained in ELISA and binding kinetics (k a , k d , and K D ) obtained through Biacore studies for each ADP are shown.

Article Snippet: As negative and positive controls, 1 μg/mL normal mouse IgG and goat anti-mouse ADAM8 Ab (AF1031, R&D Systems, RRID: AB_354549), respectively, were analyzed.

Techniques: Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: ADPs bind to five epitope clusters on ADAM8. Diagram indicating the epitope clusters for ADP binding on human ADAM8, and their partial overlap identified based on epitope binning using competitive ELISA.

Article Snippet: As negative and positive controls, 1 μg/mL normal mouse IgG and goat anti-mouse ADAM8 Ab (AF1031, R&D Systems, RRID: AB_354549), respectively, were analyzed.

Techniques: Binding Assay, Competitive ELISA

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: ADPs have potent in vitro dual metalloprotease and disintegrin (MP and DI) domain inhibitory activity. ( A ) ADAM8 MP activity was assessed in the presence of each ADP vs. its isotype-matched control IgG by measuring the release of soluble CD23 from the surface of HEK293 cells ectopically overexpressing both ADAM8 and CD23. After overnight antibody (Ab) treatment, conditioned cell media was tested for cleaved CD23 via the detection of its HA-tag in immunoblotting; images were quantified using densitometry. ( B ) ADAM8 DI activity was evaluated in the presence of each ADP vs. control IgG in assays measuring binding of α9β1-Integrin-expressing CHO cells to plates coated with recombinant human ADAM8 (rHuADAM8). Mean MP and DI activity level ± standard deviation (S.D.) from 3 independent experiments is graphed in A and B, respectively. The dashed line represents the level of activity in the presence of MAB1031. Ab-mediated percent inhibition of activity in each case was calculated as a decrease from control IgG levels, which were set to 100%. Mean percent inhibition for each ADP and for MAB1031 ± S.D. is given in red over the respective activity bars.

Article Snippet: As negative and positive controls, 1 μg/mL normal mouse IgG and goat anti-mouse ADAM8 Ab (AF1031, R&D Systems, RRID: AB_354549), respectively, were analyzed.

Techniques: In Vitro, Activity Assay, Control, Western Blot, Binding Assay, Expressing, Recombinant, Standard Deviation, Inhibition

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: ADPs with in vivo anti-tumor activity bind to the ADAM8 DI. ( A ) ADP2, ADP3, or ADP13 binding to HEK293 cells expressing full-length ADAM8 (HEK-A8) vs. remnant form ADAM8 (HEK-REM) was assessed by flow cytometry; HEK293 cells expressing empty vector DNA (HEK-EV) were used as a negative control. Representative histograms of three independent runs are shown. ( B ) Schematic representation of the ADAM8 constructs used in part A, with domain information, amino acid (AA) numbers, and immunogen used for ADP generation indicated. The broad epitope region for ADP2, ADP3, and ADP13 binding to ADAM8, identified by the flow cytometry analysis in part A, is indicated (striped box). ADAM8 domains: PRO—prodomain; MP—metalloproteinase; DI—disintegrin; CRD—cysteine-rich; ELD—EGF-like; TM—transmembrane; CYTO—cytoplasmic. ( C ) Three-dimensional model of the predicted ADAM8 extracellular structure (residues 195-647, including MP, DI, CRD, and ELD) using the crystal structure of ADAM22 as template and Swiss-model software (2003). Regions of ADP2, ADP3, and ADP13 binding, including overlapping sequences, identified through hydrogen/deuterium exchange–mass spectrometry (HDX-MS) analysis are indicated. MP with active catalytic site, DI with integrin-binding region, and hypervariable region (HVR) of CDR are shown.

Article Snippet: As negative and positive controls, 1 μg/mL normal mouse IgG and goat anti-mouse ADAM8 Ab (AF1031, R&D Systems, RRID: AB_354549), respectively, were analyzed.

Techniques: In Vivo, Activity Assay, Binding Assay, Expressing, Flow Cytometry, Plasmid Preparation, Negative Control, Construct, Software, Mass Spectrometry

Journal: Pharmaceutics

Article Title: A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer

doi: 10.3390/pharmaceutics16040536

Figure Lengend Snippet: Amino acids (AAs) within the ADAM8 DI mediating ADP2 and ADP13 binding. ( A ) AA residues important for ADP2 and ADP13 binding to ADAM8 were identified using alanine (ALA) scanning mutagenesis plus flow cytometry. Mean binding reactivity (in duplicate samples) of ADP2 or ADP13 antigen−binding fragments (Fabs) to ADAM8 protein mutated at the indicated residues (mutation) within the MP and DI vs. binding of a positive control ADAM8 Ab (Control A8 Ab), which binds outside the MP and DI regions and is therefore unaffected, is presented as a percentage of binding to wild−type (WT) ADAM8. The range of binding reactivity (maximum–minimum) in each case is indicated in parentheses. AAs identified as critical for binding (i.e., those for which Control A8 Ab binding was >70% of WT but test Ab binding was <20% of WT binding) are shown in red boxes. Blue boxes show residues of secondary importance, i.e., AAs in close proximity to critical residues whose mutation led to a substantial (although not <20% of WT) reduction in binding. Epitope AAs for ADP2 ( B ) and ADP13 ( C ) Fab binding, identified through mutagenesis, are indicated on a crystal structure model of the ADAM8 ectodomain based on the structure of vascular apoptosis−inducing protein−1.

Article Snippet: As negative and positive controls, 1 μg/mL normal mouse IgG and goat anti-mouse ADAM8 Ab (AF1031, R&D Systems, RRID: AB_354549), respectively, were analyzed.

Techniques: Binding Assay, Mutagenesis, Flow Cytometry, Positive Control, Control