Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A virus-like particle-based anti-nerve growth factor vaccine reduces inflammatory hyperalgesia: potential long-term therapy for chronic pain.

doi: 10.4049/jimmunol.1000030

Figure Lengend Snippet: FIGURE 1. Production and functional analysis of the rNGFQb vaccine. A, Production of rNGF. Murine NGF was expressed in HEK293T cells. Antipentahistidine immunoblot analysis of HEK293T lysates (first lane) and cell-culture supernatants (second lane) revealed expression of the NGF proprotein by HEK293T cells and secretion of the processed 14.7-kDa mature form of NGF into the cell-culture supernatant. Coomassie staining of NGF purified from supernatants by affinity chromatography confirmed homogeneity (third lane). B, Recognition of NGF by anti-NGF Abs. Recombinantly expressed NGF (white circles) was compared with ex vivo- purified NGF (black squares). Different dilutions of both proteins were applied to ELISA plates coated with anti-NGF mAb. Bound NGF was detected with polyclonal anti-NGF Abs. Averages of triplicates 6 SEM. C, NGF receptor binding. The NGF-specific receptor TrkA was coated onto ELISA plates and descending concentrations of both proteins applied. Receptor bound NGF was detected with polyclonal NGF Abs as in B. Averages of triplicates 6 SEM. D, Bioactivity of NGF. The NGF re- sponsive cell line TF-1 was stimulated with descending concentrations of NGF and proliferation determined by BrdU incorporation during DNA synthesis. Averages of triplicates 6 SEM. E, Analysis of NGFQb vaccine. NGF was cross-linked to VLPs with a heterobifunctional cross-linker. An immunoblot of NGF (left lane) or the NGFQb conjugate vaccine (right lane) analyzed with an anti-pentahistidine–specific Ab showed a dominant band appearing at 29 kDa corresponding to a Qb monomer (14.2 kDa) conjugated to one molecule NGF (14.7 kDa). Higher molecular mass bands correspond to Qb-oligomers linked to one NGF molecule. F, Binding of TrkA to NGFQb. Ascending concentrations of Qb (filled squares) or NGFQb (open circles) were immobilized on an ELISA plate coated with anti-Qb mAb. Bound vaccine was incubated with TrkA re- ceptor containing a human Fc domain. Binding of receptor to NGF dis- played on VLP was detected with peroxidase-conjugated goat anti-human Abs. Averages of triplicates 6 SEM. LY, lysates; SN, supernatants.

Article Snippet: Then, plates were washed six times with PBS-T and incubated with a 1:500 dilution of a sheep anti-NGF polyclonal Ab (AbD Serotec) for 1 h at RT.

Techniques: Functional Assay, Western Blot, Cell Culture, Expressing, Staining, Chromatography, Ex Vivo, Enzyme-linked Immunosorbent Assay, Binding Assay, BrdU Incorporation Assay, DNA Synthesis, Incubation