METHODS FOR BINDING BIOLOGICALLY ACTIVE MOLECULES TO SURFACES

Abstract

The invention relates to methods for binding biologically active molecules to surfaces by coating the surface with a supported reagent in a first step and then covalently coupling said biologically active molecules to the supported reagent using a biorthogonal cycloaddition reaction. Fields of application of the invention include biochemical research, medical diagnostics and the pharmaceutical industry.

Claims

1-15. (canceled)

16. Method for binding biologically active molecules to a surface of a substrate, comprising the steps of: (i) providing a carrier molecule comprising at least one click functional group, wherein the carrier molecule comprises a macromolecule selected from polypeptides, proteins, glycoproteins and carbohydrates and has a molecular weight of ≧10 kDA, (ii) binding the carrier molecule to a surface of a substrate, wherein the carrier molecule is adsorptively bound to the surface of the substrate, (iii) binding a biologically active molecule comprising at least one click functional group that is reactive towards the click functional group of the carrier molecule to the carrier molecule by cycloaddition reaction between the mutually reactive click functional groups.

17. Method according to claim 16, wherein the click functional groups are selected from the group consisting of azide, alkene, alkyne, nitrone, nitrilimine, nitrile oxide, isonitrile, tetrazole and tetrazine groups.

18. Method according to claim 16, wherein the substrate comprises plastics material, in particular polystyrene, polypropylene, polyethylene, polyamide, polymethacrylate, polycarbonate, polyacrylate or copolymers of polystyrene, glass, ceramics, latex, metal, in particular gold, and/or carbon.

19. Method according to claim 16, wherein the substrate is in the form of a plate, in particular a microplate, or in the form of microparticles or nanoparticles.

20. Method according to claim 16, wherein the carrier molecule comprises human serum albumin or bovine serum albumin.

21. Method according to claim 16, wherein the biologically active molecule is selected from the group consisting of proteins, enzymes, antibodies, nucleic acids and low molecular weight ligands of proteins, enzymes, antibodies or nucleic acids.

22. Article, comprising (i) a substrate having a surface, and (ii) a carrier molecule, wherein the carrier molecule comprises a macromolecule selected from the group consisting of polypeptides, proteins, glycoproteins and carbohydrates and has a molecular weight of ≧10 kDA and wherein the carrier molecule is adsorptively bound to the surface of the substrate and comprises at least one click functional group.

23. Article, comprising (i) a substrate having a surface, (ii) a carrier molecule, and (iii) a biologically active molecule, wherein the carrier molecule comprises a macromolecule selected from the group consisting of polypeptides, proteins, glycoproteins and carbohydrates and has a molecular weight of ≧10 kDA and wherein the carrier molecule is adsorptively bound to the surface of the substrate, and the biologically active molecule is bound to the carrier molecule via a linker which contains a group formed by click reaction.

24. Article according to claim 23, wherein the group formed by click reaction is selected from 5-membered or 6-membered heterocycles, in particular 1,2,3-triazole.

25. Article according to claim 22, wherein the carrier molecule is cross-linked or is not cross-linked.

26. Kit, comprising (i) a substrate having a surface, (ii) a carrier molecule which is adsorptively bound to the surface of the substrate and has at least one click functional group, wherein the carrier molecule comprises a macromolecule selected from the group consisting of polypeptides, proteins, glycoproteins and carbohydrates and has a molecular weight of ≧10 kDA and (iii) a reagent which allows a biologically active molecule to be modified with a click functional group which is reactive towards the click functional group of the carrier molecule (ii).

27. A carrier molecule which comprises at least one click functional group and a macromolecule selected from the group consisting of polypeptides, proteins, glycoproteins and carbohydrates, wherein said carrier molecule has a molecular weight of ≧10 kDa.

28. A method for determining an analyte in a sample, comprising binding a biologically active molecule to a surface of a substrate, wherein said substrate is contained in an article according to claim 22.

29. The carrier molecule according to claim 27, wherein said macromolecule is human serum albumin or bovine serum albumin.

30. The carrier molecule according to claim 27, wherein said carrier molecule is adsorptively or covalently bound to a surface of a substrate of an article, wherein said carrier molecule comprises at least one click functional group.

31. The carrier molecule according to claim 27, wherein said carrier molecule comprises at least one click functional group and is adsorptively or covalently bound to a surface of a substrate, and wherein said carrier molecule is part of a kit comprising (i) a substrate having a surface, (ii) a carrier molecule, and (iii) a reagent which allows a biologically active molecule to be modified with a click functional group which is reactive towards the click functional group of the carrier molecule (ii).

Description

DESCRIPTION OF THE FIGURES

[0086] FIG. 1:

[0087] Determination of PR3 autoantibodies in human serum on microplates coated with DBCO-BSA-PR3. Dilutions of a human serum with PR3 autoantibodies in the indicated concentrations were tested in microplate wells coated with DBCO-BSA-PR3 and, for comparison, in microplate wells coated with antigen-free DBCO-BSA. The binding of PR3 autoantibodies to the well surface was thereby determined by means of a peroxidase-labelled anti-human IgG antibody and a TMB colour reaction by measuring the optical density at 450 nm.

[0088] FIG. 2:

[0089] Determination of MPO autoantibodies in human serum on microplates coated with DBCO-BSA-MPO. Dilutions of a human serum with MPO autoantibodies in the indicated concentrations were tested in microplate wells coated with DBCO-BSA-MPO and, for comparison, in microplate wells coated with antigen-free DBCO-BSA. The binding of MPO autoantibodies to the well surface was thereby determined by means of a peroxidase-labelled anti-human IgG antibody and a TMB colour reaction by measuring the optical density at 450 nm.

[0090] FIG. 3:

[0091] Determination of PR3 autoantibodies in human serum on paramagnetic nanoparticles coated with DBCO-BSA-PR3. Paramagnetic nanoparticles coated with -BSA-PR3 were added to dilutions of a human serum with PR3 autoantibodies in the indicated concentrations. The binding of PR3 autoantibodies to the nanoparticles was thereby determined by means of a peroxidase-labelled anti-human IgG antibody and a TMB colour reaction by measuring the optical density at 450 nm.

[0092] FIG. 4:

[0093] Determination of cardiolipin/β2-glycoprotein autoantibodies in human serum on paramagnetic nanoparticles coated with azide-BSA-β2-glycoprotein or with azide-BSA-cardiolipin-β2-glycoprotein. Coated paramagnetic nanoparticles were added to dilutions of a human serum with cardiolipin/β2-glycoprotein autoantibodies in the indicated concentrations. The binding of cardiolipin/β2-glycoprotein autoantibodies to the nanoparticles was thereby