Method for immobilizing glycoproteins, constructs and apparatuses made thereby

• US 20050003557A1
• Filed: 03/05/2004
• Published: 01/06/2005
• Est. Priority Date: 03/06/2003
• Status: Abandoned Application

• Current Assignee: Micronas Holding GmbH (TDK Corporation)
• Original Assignee: Micronas Holding GmbH (TDK Corporation)
• Inventors: Klapproth, Holger
• Application Number:
  US10/793,323
• Publication Number:
  US 20050003557A1
• Time in Patent Office: Days
• US Class Current: 436/518
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

A method for covalently immobilizing glycoproteins such as immunoglobulins on solid, semi-solid or gel-like surfaces, and constructs suitable therefor, as well as surfaces and devices that comprise correspondingly immobilized glycoproteins.

Claims

1. A construct, comprising:

a heterobifunctional cross-linker molecule having a spacer region having a section for non-covalent adsorption onto a surface; and

a protein selected from the group consisting of a glycoprotein and a functional fragment of a glycoprotein.

2. A construct according to claim 1, wherein said section for non-covalent adsorption is selected from the group consisting of a hydrophobic section, a positively-charged hydrophilic section, and a negatively-charged hydrophilic section.

3. A construct according to claim 2, wherein said spacer region comprises a hydrophilic section and a hydrophobic section.

4. A construct according to claim 1, wherein said cross-linker molecule comprises at least one photoreactive group for covalent coupling with said surface.

5. A construct according to claim 4, wherein said at least one photoreactive group is disposed at a position selected from the group consisting of an end of said spacer, within said spacer, and both within and at an end of said spacer.

6. A construct according to claim 4, wherein said at least one photoreactive group is selected from the group consisting of arylazides, benzophenones, benzothiones, anthraquinones, anthrathiones, thymidine, and derivatives thereof.

7. A construct according to claim 1, wherein said cross-linker molecule is covalently coupled with said glycoprotein by a carbohydrate component of said glycoprotein.

8. A construct according to claim 1, wherein said cross-linker molecule is coupled with said functional fragment of the glycoprotein by a thiol group of said glycoprotein.

9. A construct according to claim 1, wherein said glycoprotein is an antibody.

10. A construct according to claim 1, wherein said functional fragment of a glycoprotein is selected from the group consisting of a FAB-fragment, F(ab′

)₂ fragment, and a SCAB protein.

11. A construct according to claim 1, wherein said glycoprotein or functional fragment thereof is of recombinant origin.

12. A surface having a construct according to claims 1 covalently immobilized thereon.

13. A surface according to claim 12, comprising a plurality of constructs.

14. A surface according to claim 12, wherein the surface comprises a material selected from the group consisting of glass, quartz glass, quartz, silicon, polymers, including PMMA, polystyrene, polyethylene, polypropylene and PVC, membranes including nitrocellulose, nylon and microfibers, and paper.

15. An analytical or diagnostic apparatus comprising a construct according to claim 1.

16. An analytical or diagnostic device according to claim 15 having a structure selected from the group consisting of a biochip, a sensor chip, a reaction column and a microtiter plate.

25. A method according to claim 24, wherein the construct is a construct according to claim 1.

27. An analytical or diagnostic device comprising a surface according to claim 12.

17. A method for producing a construct comprising a glycoprotein or functional fragment thereof and a heterobifunctional cross-linker molecule having a spacer region, comprising the following steps:

(a) incubating a protein selected from the group consisting of a glycoprotein and a functional fragment thereof under conditions suitable for production of a chemical group selected from the group consisting of aldehyde groups and thiol groups, to produce a product;

(b) covalently coupling the product of step (a) with a cross-linker molecule by a chemical group of the cross-linker selected from the group consisting of an amino group and a maleinimide group, to produce a construct;

as well as, optionally,

(c) purifying or isolating the construct.

18. A method according to claim 17, wherein said cross-linker molecule comprises at least one photoreactive group as a second functional group and wherein the spacer region has a section for non-covalent adsorption onto a surface.

19. A method according to claim 18, wherein said at least one photoreactive group is disposed at a position selected from the group consisting of an end of said spacer, within said spacer, and both within and at an end of said spacer.

20. A construct comprising a glycoprotein or functional fragment thereof and a heterobifunctional cross-linker molecule having a spacer region having a section for non-covalent adsorption on a surface, obtainable by the method of claim 17.

21. A construct according to claim 20, characterized in that the glycoprotein is an antibody.

22. A construct according to claim 20, characterized in that the functional fragment of the glycoprotein selected from the group consisting of a FAB fragment, F(ab′

)₂ fragment, and a SCAB protein.

23. A method, comprising the steps of:

providing a construct according to claim 20;

and

incorporating the construct into an analytical or diagnostic apparatus, wherein the apparatus is selected from the group consisting of a biochip, a sensor chip, a reaction column and a microtitre plate.

26. A method according to claim 24, wherein the construct is a construct according to claim 20.

28. An analytical or diagnostic device according to claim 17 having a structure selected from the group consisting of a biochip, a sensor chip, a reaction column and microtiter plate.

24. A process for covalent immobilization of a glycoprotein or functional fragment thereof on a surface with use of a heterobifunctional cross-linker molecule, comprising the following steps:

(a) applying to a surface a construct of a glycoprotein or functional fragment thereof and a heterobifunctional cross-linker molecule having a spacer region and at least one photoreactive group; and

(b) immobilizing the construct on the surface by irradiation of a contact point with light of a suitable wavelength.