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Functional neutralization of NGF binding to the TrkA receptor and SP-induced mast cell degranulation by antibodies elicited by rNGFSP in dogs. A) Inhibition of NGF–TrkA binding. Neutralization of NGF binding to the TrkA receptor was assessed by ELISA. Biotinylated NGF was incubated with TrkA in the presence of pooled sera from non-immunized dogs at day 0 (D0), sera from rNGFSP-immunized dogs at day 56 (D56), or a commercial monoclonal anti-NGF antibody (0.5 µg/mL). NGF alone served as a positive control. Sera were diluted 1:10 prior to the assay.. * p < 0.05. B) Inhibition of SP-induced mast cell degranulation. Flow cytometry analysis of mast cells stimulated with SP conjugated to BSA in the presence of IgG from non-immunized (D0) or immunized (D56) dogs. Representative plots show <t>avidin–FITC-positive</t> degranulating cells. The percentage of degranulating mast cells from replicate experiments is summarized on the right. All data are expressed as mean ± SEM, with * p < 0.05 compared to Day 0, determined by two-way ANOVA followed by Tukey’s multiple comparisons test. C) Confocal microscopy of mast cell degranulation. Mast cells were stained with avidin–FITC (green) and DAPI (blue). SP or the positive control compound 48/80 (10 µM) induced granule release, whereas IgG from immunized dogs (D56) reduced SP-induced degranulation. Scale bar: 10 µm.
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Functional neutralization of NGF binding to the TrkA receptor and SP-induced mast cell degranulation by antibodies elicited by rNGFSP in dogs. A) Inhibition of NGF–TrkA binding. Neutralization of NGF binding to the TrkA receptor was assessed by ELISA. Biotinylated NGF was incubated with TrkA in the presence of pooled sera from non-immunized dogs at day 0 (D0), sera from rNGFSP-immunized dogs at day 56 (D56), or a commercial monoclonal anti-NGF antibody (0.5 µg/mL). NGF alone served as a positive control. Sera were diluted 1:10 prior to the assay.. * p < 0.05. B) Inhibition of SP-induced mast cell degranulation. Flow cytometry analysis of mast cells stimulated with SP conjugated to BSA in the presence of IgG from non-immunized (D0) or immunized (D56) dogs. Representative plots show <t>avidin–FITC-positive</t> degranulating cells. The percentage of degranulating mast cells from replicate experiments is summarized on the right. All data are expressed as mean ± SEM, with * p < 0.05 compared to Day 0, determined by two-way ANOVA followed by Tukey’s multiple comparisons test. C) Confocal microscopy of mast cell degranulation. Mast cells were stained with avidin–FITC (green) and DAPI (blue). SP or the positive control compound 48/80 (10 µM) induced granule release, whereas IgG from immunized dogs (D56) reduced SP-induced degranulation. Scale bar: 10 µm.
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Functional neutralization of NGF binding to the TrkA receptor and SP-induced mast cell degranulation by antibodies elicited by rNGFSP in dogs. A) Inhibition of NGF–TrkA binding. Neutralization of NGF binding to the TrkA receptor was assessed by ELISA. Biotinylated NGF was incubated with TrkA in the presence of pooled sera from non-immunized dogs at day 0 (D0), sera from rNGFSP-immunized dogs at day 56 (D56), or a commercial monoclonal anti-NGF antibody (0.5 µg/mL). NGF alone served as a positive control. Sera were diluted 1:10 prior to the assay.. * p < 0.05. B) Inhibition of SP-induced mast cell degranulation. Flow cytometry analysis of mast cells stimulated with SP conjugated to BSA in the presence of IgG from non-immunized (D0) or immunized (D56) dogs. Representative plots show <t>avidin–FITC-positive</t> degranulating cells. The percentage of degranulating mast cells from replicate experiments is summarized on the right. All data are expressed as mean ± SEM, with * p < 0.05 compared to Day 0, determined by two-way ANOVA followed by Tukey’s multiple comparisons test. C) Confocal microscopy of mast cell degranulation. Mast cells were stained with avidin–FITC (green) and DAPI (blue). SP or the positive control compound 48/80 (10 µM) induced granule release, whereas IgG from immunized dogs (D56) reduced SP-induced degranulation. Scale bar: 10 µm.
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Functional neutralization of NGF binding to the TrkA receptor and SP-induced mast cell degranulation by antibodies elicited by rNGFSP in dogs. A) Inhibition of NGF–TrkA binding. Neutralization of NGF binding to the TrkA receptor was assessed by ELISA. Biotinylated NGF was incubated with TrkA in the presence of pooled sera from non-immunized dogs at day 0 (D0), sera from rNGFSP-immunized dogs at day 56 (D56), or a commercial monoclonal anti-NGF antibody (0.5 µg/mL). NGF alone served as a positive control. Sera were diluted 1:10 prior to the assay.. * p < 0.05. B) Inhibition of SP-induced mast cell degranulation. Flow cytometry analysis of mast cells stimulated with SP conjugated to BSA in the presence of IgG from non-immunized (D0) or immunized (D56) dogs. Representative plots show avidin–FITC-positive degranulating cells. The percentage of degranulating mast cells from replicate experiments is summarized on the right. All data are expressed as mean ± SEM, with * p < 0.05 compared to Day 0, determined by two-way ANOVA followed by Tukey’s multiple comparisons test. C) Confocal microscopy of mast cell degranulation. Mast cells were stained with avidin–FITC (green) and DAPI (blue). SP or the positive control compound 48/80 (10 µM) induced granule release, whereas IgG from immunized dogs (D56) reduced SP-induced degranulation. Scale bar: 10 µm.

Journal: Biotechnology Reports

Article Title: Production and characterization of rNGFSP: a recombinant fusion immunogen eliciting dual anti-NGF and anti-Substance P therapeutic antibodies for Degenerative Joint Disease

doi: 10.1016/j.btre.2026.e00946

Figure Lengend Snippet: Functional neutralization of NGF binding to the TrkA receptor and SP-induced mast cell degranulation by antibodies elicited by rNGFSP in dogs. A) Inhibition of NGF–TrkA binding. Neutralization of NGF binding to the TrkA receptor was assessed by ELISA. Biotinylated NGF was incubated with TrkA in the presence of pooled sera from non-immunized dogs at day 0 (D0), sera from rNGFSP-immunized dogs at day 56 (D56), or a commercial monoclonal anti-NGF antibody (0.5 µg/mL). NGF alone served as a positive control. Sera were diluted 1:10 prior to the assay.. * p < 0.05. B) Inhibition of SP-induced mast cell degranulation. Flow cytometry analysis of mast cells stimulated with SP conjugated to BSA in the presence of IgG from non-immunized (D0) or immunized (D56) dogs. Representative plots show avidin–FITC-positive degranulating cells. The percentage of degranulating mast cells from replicate experiments is summarized on the right. All data are expressed as mean ± SEM, with * p < 0.05 compared to Day 0, determined by two-way ANOVA followed by Tukey’s multiple comparisons test. C) Confocal microscopy of mast cell degranulation. Mast cells were stained with avidin–FITC (green) and DAPI (blue). SP or the positive control compound 48/80 (10 µM) induced granule release, whereas IgG from immunized dogs (D56) reduced SP-induced degranulation. Scale bar: 10 µm.

Article Snippet: After stimulation, cells were centrifuged (1000 × g, 10 min, 4 °C), resuspended in 150 μL PBS, and stained with 2 μg/mL Avidin-FITC (Thermo Scientific, #A821) for 20 min at room temperature.

Techniques: Functional Assay, Neutralization, Binding Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, Flow Cytometry, Avidin-Biotin Assay, Confocal Microscopy, Staining