Biosensors, method for obtaining the same and uses thereof

Abstract

The invention relates to biosensors, methods for obtaining them and their use for detecting, assaying or locating, in direct immunofluorescence, a ligand such as an antigen or hapten, in a heterogeneous population. The biosensor includes (i) at least one fragment of a receptor which is protein in nature, capable of binding to a ligand via an active site, where at least one amino acid residues of the fragment located in the proximity of the active site is naturally present in the form of a cystein (Cys) residue, or is substituted with a Cys residue, and (ii) a fluorophore coupled to the Cys residue.

1 - 32 . (canceled) 
     
     
         33 . A biosensor, consisting of (i) at least one fragment of a receptor which is a protein, wherein said receptor fragment is capable of binding to a suitable ligand via an active site to form a receptor fragment-ligand complex, and in which receptor fragment at least one of its amino acid residues located in the proximity of said active site is naturally present in the form of a Cys residue, or is substituted with a Cys residue, and (ii) a fluorophore coupled to said Cys residue(s). 
     
     
         34 . The biosensor as claimed in  claim 33 , wherein said Cys residue in the receptor fragment-ligand complex: (i) is in direct contact with the ligand, or (ii) is in contact via a water molecule with the ligand, or (iii) has a solvent accessible surface area (ASA) which is modified by binding of the ligand, the solvent being defined by spheres with a radius comprised in a range from 1.4 to 30 Å. 
     
     
         35 . The biosensor as claimed in  claim 34 , wherein the Cys residue has a solvent accessible surface area (ASA) which is modified by binding of the ligand, the solvent being defined by spheres with a radius comprised in a range from 1.4 to 2.9 Å. 
     
     
         36 . The biosensor as claimed in  claim 34 , wherein the atom in the γ position or in the δ position of said Cys residue in the free receptor, is accessible to a solvent defined by spheres with a radius of 1.4 Å. 
     
     
         37 . The biosensor as claimed in  claim 33 , wherein said receptor is an antibody or an antibody fragment. 
     
     
         38 . The biosensor as claimed in  claim 37 , wherein the receptor is a natural or artificial monoclonal antibody. 
     
     
         39 . The biosensor as claimed in  claim 33 , wherein the fluorophore is selected from the group consisting of: IANBD, CNBD, acrylodan, 5-iodoacetamidofluorescein and a fluorophore having an aliphatic chain of 1 to 6 carbon atoms. 
     
     
         40 . The biosensor as claimed in  claim 33 , wherein the biosensor is in soluble form. 
     
     
         41 . The biosensor as claimed in  claim 33 , wherein the biosensor is immobilized on a suitable solid support. 
     
     
         42 . The biosensor as claimed in  claim 41 , wherein the solid support is selected from the group consisting of microplates and optical fibers. 
     
     
         43 . A protein-based chip, comprising a solid support, on which at least one biosensor as claimed in  claim 33  is immobilized. 
     
     
         44 . A reagent comprising at least one biosensor as claimed in  claim 33 . 
     
     
         45 . A method for detecting, assaying or locating a ligand in an heterogeneous sample, comprising: bringing said heterogeneous sample into contact with at least one reagent as claimed in  claim 44 , and a detection step for detecting, assaying or locating a ligand in a heterogeneous sample. 
     
     
         46 . A kit for detecting, assaying or locating ligands, comprising at least one reagent as claimed in  claim 44 . 
     
     
         47 . A kit for screening for inhibitors of the ligand/receptor fragment interaction, comprising at least one reagent as claimed in  claim 44 .