Background Tumor necrosis factor (TNF) plays an essential function in host defense. On the other hand, its overexpression may be deleterious and lead to autoimmune disorders. Therapeutic inhibition of TNF (mostly by recombinant anti-TNF antibodies) is effective in rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease and other autoimmune disorders. In spite of this success, detailed knowledge concerning sites and dynamics of TNF overexpression in the course of developing disease, as well as in the course of treatment is lacking.
Most of the current methods of TNF detection in tissues of experimental laboratory animals are invasive, and they may themselves affect the course of the disease. A molecular probe, which could help to visualize TNF production in vivo, would be useful.
Objectives To generate a fluorescent sensor of TNF which would specifically accumulate at the sites of its expression and can be used for in vivo imaging. To test such sensor in various autoimmune disease models in mice.
Methods Single domain antibodies with high affinity to TNF were selected by phage display from immunized Bactrian camel1. They were genetically fused to far-red fluorescent protein Katushka2. The resultant fluorescent sensor of TNF was expressed in E.Coli (Bl21) and purified from bacterial lysates by IMAC on Ni-NTA resin. Protein concentration was measured by BCA assay, sample purity was assessed by SDS-PAGE. Multimerization in native confirmation was detected by HPLC. Binding to TNF was measured by ELISA and SPR. TNF inhibitory properties of fluorescent sensor of TNF were assessed in vitro by cytotoxic assay on L929 fibroblasts and in vivo in LPS/DgalN toxicity model on C57Bl6 mice. In vivo fluorescence in Collagen Induced Arthritis (CIA) model was measured using Carestream FX Pro In-vivo imaging system. Ex-vivo fluorescence in ConA induced hepatitis and CIA were detected by confocal microscopy.
Results We generated, expressed and purified fluorescent sensor of TNF, which is a fusion protein comprising anti-TNF recombinant single domain antibody and the far-red fluorescent protein Katushka. This fluorescent sensor can be visualized in-vivo, as it specifically accumulates in inflamed tissues during model pathologies. This fusion protein binds TNF with high affinity but does not interfere with simultaneous TNF receptor binding and does not inhibit TNF biological activity. It can be used to highlight sites of TNF overexpression in model diseases.
Conclusions We have generated a novel type of molecular probe for in vivo imaging of TNF in autoimmune pathologies which may be applied for elucidation the patterns of TNF expressions during the onset and acute phase of disease, as well as during its treatment.
Efimov, G. A., Kruglov, A. A., Tillib, S. V., Kuprash, D. V., & Nedospasov, S. A (2009). Tumor Necrosis Factor and the consequences of its ablation in vivo. Molecular immunology, 47(1), 19–27.
Shcherbo, D., Merzlyak, E., Chepurnykh, T., Fradkov, A., Ermakova, G., Solovieva, E., Lukyanov, K., et al. (2007). Bright far-red fluorescent protein for whole-body imaging. Nature Methods, 4(9), 741–746.
Disclosure of Interest None Declared
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