rabbit anti canine igg horseradish peroxidase  (Novus Biologicals)

Journal: iScience

Article Title: Development of OX40 agonists for canine cancer immunotherapy

doi: 10.1016/j.isci.2022.105158

Figure Lengend Snippet: Development and characterization of cFcOX40L B and cFcOX40L D (A) Expression and purification of cFcOX40L B . Western blot analysis of conditioned media. The cFcOX40L B was expressed in ExpiCHO-S cells. The conditioned media was resolved under reducing and nonreducing conditions, and the cFcOX40L B was detected using an anti-IgG Fc antibody. The cFcOX40L B , as expected, forms dimers of ∼101 kDa under nonreducing conditions. (R-reducing condition, NR-nonreducing). (B) Purity of cFcOX40L B assessed by SDS-PAGE. The cFcOX40L B was expressed and purified from the ExpiCHO-S cells by affinity (Protein A) and size-exclusion chromatography. The purified cFcOX40L B was resolved under reducing (R) condition and stained with GelCode Blue Stain. (C) cFcOX40L B binds to canine OX40. 293F-OX40 cells were stained with cFcOX40L B , washed, and bound cFcOX40L B was detected using anti-Fc-750 antibody by flow cytometry. (D) cFcOX40L B does not bind to untransfected 293F cells. (E) Expression and purification of cFcOX40L D . Western blot analysis of conditioned media. The cFcOX40L D was expressed in ExpiCHO-S cells. The conditioned media was resolved under reducing and nonreducing conditions, and the cFcOX40L D was detected using an anti-IgG Fc antibody. The cFcOX40L D , as expected, forms dimers of ∼101 kDa under nonreducing conditions. (R-reducing condition, NR-nonreducing). (F) Purity of cFcOX40L D assessed by SDS-PAGE. The cFcOX40L D was purified by affinity (StrepTrap HP column) and size-exclusion chromatography. The purified cFcOX40L D was resolved under reducing (R) and nonreducing (NR) conditions and stained with GelCode Blue Stain. (G) cFcOX40L D binds to canine OX40. 293F-OX40 cells were stained with cFcOX40L D , washed, and bound cFcOX40L D was detected using anti-Fc-DyLight 750 antibody by flow cytometry. (H) cFcOX40L D does not bind to untransfected 293F cells.

Article Snippet: Rabbit anti-canine IgG Fc antibodies (Novus Biologicals) and anti-Strep Tag II antibodies were used to detect recombinant proteins.

Techniques: Expressing, Purification, Western Blot, SDS Page, Size-exclusion Chromatography, Staining, Flow Cytometry

Journal: iScience

Article Title: Development of OX40 agonists for canine cancer immunotherapy

doi: 10.1016/j.isci.2022.105158

Figure Lengend Snippet: Activated canine CD3 T cells express OX40 (A and B) CD4 and CD8 T cells were stimulated with concanavalin A (5 μg/mL) for 72 h. Activated cPBMCs were stained with cFcOX40 L B fusion protein, and the bound proteins were detected using goat anti-canine IgG Fc DyLight-750 Ab. cPBMCs were also stained with anti-CD3, anti-CD4, and anti-CD8 mAbs. The CD3 T cells were gated to obtain CD4 and CD8 populations, and cOX40 expression was analyzed on these immune subsets by flow cytometry. Canine IgG was used as an isotype control. cOX40 is predominantly expressed on helper T cells, while its expression is restricted to a small population of cytotoxic T cells. (C) ConA induces OX40 expression on canine Tregs. Activated cPBMCs were similarly stained with anti-CD4CD3, CD8, and cFcOX40L B fusion protein. The stained cPBMCs were fixed, permeabilized, and treated with anti-FoxP3 eFluor 450 Ab. The CD4 + CD3 + T cell population was gated for FoxP3 expression and analyzed for OX40 expression. ConA stimulation induces the expression of cOX40 on cTregs. The binding of cFcOX40L B to various T-lymphocyte subsets is shown. The cFcOX40L D also binds similarly to cOX40 on T-lymphocytes (data not shown). (D) cPBMCs from three healthy dogs were isolated, stimulated with ConA, and analyzed for cOX40 expression. The cOX40 expression on CD4 + CD3 + , CD8 + CD3 + , and CD3 + CD4 + FoxP3 + T cells from three healthy dogs is shown in the bar graph. Data are represented as mean +/− standard deviation.

Article Snippet: Rabbit anti-canine IgG Fc antibodies (Novus Biologicals) and anti-Strep Tag II antibodies were used to detect recombinant proteins.

Techniques: Staining, Expressing, Flow Cytometry, Control, Binding Assay, Isolation, Standard Deviation

Journal: iScience

Article Title: Development of OX40 agonists for canine cancer immunotherapy

doi: 10.1016/j.isci.2022.105158

Figure Lengend Snippet: cFcOX40L B and cFcOX40L D bind and activate human OX40 (A) cFcOX40L B and cFcOX40L D bind to human OX40. OX40 effector cells were stained with cFcOX40L B and cFcOX40L D, and the bound proteins were detected using goat anti-canine IgG Fc DyLight-750 Ab. Both cFcOX40L B and cFcOX40L D successfully bind to human OX40 on OX40 effector cells. (B) cFcOX40L B and cFcOX40L D induce NF-κB promotor activity in OX40 effector cells. OX40 effector cells were induced for 5 h with a serial titration of cFcOX40L B , cFcOX40L D, and isotype control (canine IgG Fc). After 5 h of induction, luminescence was detected using Bio-Glo reagent. A three-parameter logistic regression dose-response curve was fit to calculate the EC50 of each recombinant protein.

Article Snippet: Rabbit anti-canine IgG Fc antibodies (Novus Biologicals) and anti-Strep Tag II antibodies were used to detect recombinant proteins.

Techniques: Staining, Activity Assay, Titration, Control, Recombinant

Journal: iScience

Article Title: Development of OX40 agonists for canine cancer immunotherapy

doi: 10.1016/j.isci.2022.105158

Figure Lengend Snippet:

Article Snippet: Rabbit anti-canine IgG Fc antibodies (Novus Biologicals) and anti-Strep Tag II antibodies were used to detect recombinant proteins.

Techniques: Control, Recombinant, Transfection, Staining, Enzyme-linked Immunosorbent Assay, Luciferase, Isolation, TaqMan Assay, RNA Sequencing, Bioassay, Software