Methods For Analyzing Interactions Between Proteins And Sugar Chains

• US 20070202535A1
• Filed: 12/24/2004
• Published: 08/30/2007
• Est. Priority Date: 12/25/2003
• Status: Active Grant

• Current Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan)
• Original Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan)
• Inventors: Hirabayashi, Jun, Nakamura, Sachiko, Kuno, Atsushi, Uchiyama, Noboru
• Granted Patent: US 8,008,094 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: Y10S 435/97 Test strip or test slide

Abstract

As a result of investigating the optimum conditions of methods for immobilizing proteins that interact with sugar chains onto a substrate, it was revealed that coating the surface of a slide glass with GTMS enables immobilization at a higher S/N ratio than conventionally possible. Moreover, by using a substrate to which a rubber with a number of holes was affixed to form a number of reaction vessels, and further by spotting lectins onto the substrate and washing with PBST, the weak interactions between sugar chains and lectins were successfully detected with improved sensitivity. In addition, by introducing an evanescent excitation-type scanner, it became possible to detect the interactions between lectins and sugar chains without washing away the probe solution.

Claims

1. :

A method for analyzing an interaction between a sugar chain and a protein that interacts with a sugar chain, wherein the method comprises the steps of;

(a) contacting a fluorescently labeled subject sugar chain or subject glycoconjugate with a substrate onto which a protein that interacts with a sugar chain has been immobilized; and

(b) measuring the intensity of an excited fluorescence after applying an excitation light without washing the substrate.

2. :

The method of claim 1, wherein the substrate onto which the protein that interacts with the sugar chain has been immobilized is a substrate coated with a compound comprising an epoxy group as an active group.

3. :

The method of claim 2, wherein the compound comprising an epoxy group as an active group is 3-glycidoxypropyl trimethoxysilane (GTMS).

11. :

The method of claim 1, wherein the protein that interacts with a sugar chain is a lectin, an enzymatic protein comprising a sugar-binding domain, a cytokine having an affinity for a sugar chain, or an antibody that interacts with a sugar chain.

12. :

The method of claim 1, wherein the excitation light is an evanescent wave.

13. :

The method of claim 1, wherein the glycoconjugate is a glycoprotein, a proteoglycan, or a glycolipid.

4. :

A method for analyzing an interaction between a sugar chain and a protein that interacts a with sugar chain, wherein the method comprises the steps of;

(a) contacting a protein that interacts with a fluorescently labeled sugar chain with a substrate onto which a subject glycoconjugate has been immobilized; and

(b) measuring the intensity of an excited fluorescence after applying an excitation light without washing the substrate.

5. :

The method of claim 4, wherein the substrate onto which the subject glycoconjugate has been immobilized is a substrate coated with a compound comprising an epoxy group as an active group.

6. :

The method of claim 5, wherein the compound comprising an epoxy group as an active group is 3-glycidoxypropyl trimethoxysilane (GTMS).

23. :

The method of claim 4, wherein the protein that interacts with a sugar chain is a lectin, an enzymatic protein comprising a sugar-binding domain, a cytokine having an affinity for a sugar chain, or an antibody that interacts with a sugar chain.

25. :

The method of claim 4, wherein the excitation light is an evanescent wave.

27. :

The method of claim 4, wherein the glycoconjugate is a glycoprotein, a proteoglycan, or a glycolipid.

7. :

A method for analyzing an interaction between a sugar chain and a protein that interacts with a sugar chain, wherein the method comprises the steps of;

(a) contacting a subject glycoconjugate with a substrate onto which a protein that interacts with a region other than a sugar chain of a glycoconjugate has been immobilized;

(b) contacting a fluorescently labeled protein that interacts with a sugar chain with the substrate obtained in step (a); and

(c) measuring the intensity of an excited fluorescence after applying an excitation light without washing the substrate.

8. :

The method of claim 7, wherein the substrate onto which the protein that interacts with a region other than a sugar chain of a glycoconjugate has been immobilized is a substrate coated with a compound comprising an epoxy group as an active group.

9. :

The method of claim 8, wherein the compound comprising an epoxy group as an active group is 3-glycidoxypropyl trimethoxysilane (GTMS).

10. :

The method of claim 7, wherein the protein that interacts with a region other than a sugar chain of a glycoconjugate is an antibody.

24. :

The method of claim 7, wherein the protein that interacts with a sugar chain is a lectin, an enzymatic protein comprising a sugar-binding domain, a cytokine having an affinity for a sugar chain, or an antibody that interacts with a sugar chain.

26. :

The method of claim 7, wherein the excitation light is an evanescent wave.

28. :

The method of claim 7, wherein the glycoconjugate is a glycoprotein, a proteoglycan, or a glycolipid.

14. :

A substrate coated with a compound comprising an epoxy group as an active group and onto which a protein that interacts with a sugar chain or a protein that interacts with a region other than a sugar chain of a glycoconjugate has been immobilized.

15. :

The substrate of claim 14, wherein the compound comprising an epoxy group as an active group is 3-glycidoxypropyl trimethoxysilane (GTMS).

16. :

The substrate of claim 14, wherein the protein that interacts with a region other than a sugar chain of a glycoconjugate is an antibody.

17. :

The substrate of claim 14, wherein the protein that interacts with a sugar chain is a lectin, an enzymatic protein comprising a sugar-binding domain, a cytokine having an affinity for a sugar chain, or an antibody that interacts with a sugar chain.

18. :

The substrate of claim 14, wherein the glycoconjugate is a glycoprotein, a proteoglycan, or a glycolipid.

19. :

A method for producing a substrate, wherein the method comprises the steps of;

(a) coating the substrate with a compound comprising an epoxy group as an active group; and

(b) immobilizing a protein that interacts with a sugar chain or a protein that interacts with a region other than a sugar chain of a glycoconjugate onto the substrate obtained in step (a).

20. :

The method of claim 19, wherein the protein that interacts with a region other than a sugar chain of a glycoconjugate is an antibody.

21. :

The method of claim 19, wherein the protein that interacts with a sugar chain is a lectin, an enzymatic protein comprising a sugar-binding domain, a cytokine having an affinity for a sugar chain, or an antibody that interacts with a sugar chain.

22. :

The method of claim 19, wherein the glycoconjugate is a glycoprotein, a proteoglycan, or a glycolipid.