Special Issue: Nanobody.
Chames P, Rothbauer U
Monoclonal antibodies are major tools used in various fields including proteomic approaches, diagnosis or therapy.
The specificity of this platform is to yield single domain antibodies (Nanobodies). These small and compact fragments (13 kDa) corresponding to the variable domains of a subtype of llama antibodies are characterized by outstanding properties in terms of production, stability, and their propensity to bind buried epitopes inaccessible to conventional antibodies. Most nanobodies can be expressed as soluble and active fragments without disulfide bond formation, in a reducing environment such as the cell cytoplasm or nucleus. This particularity allows their use as intracellular tool to precisely knock out a specific interaction of a target protein without interfering with other interactions of this target.
The mission of the nanobody platform is to help research teams with the isolation of specific nanobodies (single domain antibodies which can bind to buried epitopes inaccessible to conventional antibodies).
The selection and screening steps, based on phage display, can be performed on recombinant proteins, transfected cells, intact cells or lysate of cell lines. Nanobodies are produced and purified from E. coli, and can be fused to various tags (c-Myc, 6-His), modified with an extra C-terminal free cystein allowing a site-directed conjugation to nanoparticles, or biotinylated in vivo.
Genes coding for nanobodies can also be fused to the gene of a reporter molecule such as GFP, mRFP1.
The resulting constructs can be directly used as probe in living cells or for protein localization.
Homemade (proprietary) libraries
Around 15 nanobody libraries are already available. They have been built after llama immunization with:
various recombinant proteins
biopsies of various cancer types
The platform will perform llama immunization and library construction steps.
The platform can welcome researchers, technicians or trained students to perform the various steps once the project is validated.
Gradient PCR cycler, 3 laminar flow hoods for phage display, 2 laminar flow hoods for cell culture, microincubators for 15 microtiter plates, Tecan infinite M1000, Flow cytometer (3 lasers, 8 colors), AKTA purification system, 15 L fermentor.
Chames P, Rothbauer U
Chames P, Wurch T
González C, Chames P, Kerfelec B, Baty D, Robert P, Limozin L