title: Reagentless fluorescent biosensors from artificial families of antigen binding proteins.
abstract: Antibodies and artificial families of antigen binding proteins (AgBP) are constituted by a connected set of hypervariable (or randomized) residue positions, supported by a constant polypeptide backbone. The residues that form the binding site for a given antigen, are selected among the hypervariable residues. We showed that it is possible to transform any AgBP of these families into a reagentless fluorescent biosensor, specific of the target antigen, simply by coupling a solvatochromic fluorophore to one of the hypervariable residues that have little or no importance for the interaction with the antigen, after changing this residue into cysteine by mutagenesis. We validated this approach with a DARPin (Designed Ankyrin Repeat Protein) and a Nanofitin (also known as Affitin) with high success rates. Reagentless fluorescent biosensors recognize their antigen in an immediate, quantitative, selective and specific way, without any manipulation of the sample to analyze or addition of reagent.
authors.affiliation: Institut Pasteur, Department of Infection and Epidemiology, Unit of Molecular Prevention and Therapy of Human Diseases, 25 rue Docteur Roux, 75724 Paris Cedex 15, France.
authors.forename: Frederico F
authors.initials: FF
authors.lastname: Miranda
authors.forename: Elodie
authors.initials: E
authors.lastname: Brient-Litzler
authors.forename: Nora
authors.initials: N
authors.lastname: Zidane
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pecorari
authors.forename: Hugues
authors.initials: H
authors.lastname: Bedouelle
mesh_terms: D017089:Ankyrin Repeat; D000941:Antigens / Q000032:analysis*; D015374:Biosensing Techniques / Q000379:methods* / Q000706:statistics & numerical data; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008958:Models, Molecular; D016296:Mutagenesis; D015202:Protein Engineering
chemical_list: D000941:Antigens; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D003545:Cysteine