Methods for analyzing interactions between proteins and sugar chains

• US 8,008,094 B2
• Filed: 12/24/2004
• Issued: 08/30/2011
• 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
• Primary Examiner(s): Nguyen; Bao Thuy L
• Application Number:
  US10/596,692
• Publication Number:
  US 20070202535A1
• Time in Patent Office: 2,440 Days
• Field of Search: 435/7.1, 435/7.92, 435/7.94, 435/7.95
• US Class Current: 436/518
• 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 glass substrate onto which a protein that interacts with a sugar chain has been immobilized, wherein the glass substrate is coated with a compound comprising an epoxy group as an active group, and wherein a number of reaction vessels are formed on the glass substrate by affixing a rubber having a number of holes on the glass substrate;

(b) measuring the intensity of an excited fluorescence after applying an evanescent wave generated by injecting an excitation light from the edge of the glass substrate, without washing the glass substrate;

(c) digitizing the fluorescence intensity; and

(d) quantifying the fluorescence intensity.

2. The method of claim 1, wherein the compound comprising an epoxy group as an active group is 3-glycidoxypropyl trimethoxysilane (GTMS).

3. 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.

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

8. The method of claim 3, wherein the protein that interacts with a sugar chain is a lectin.

9. The method of claim 1, wherein the evanescent wave is generated by total internal reflection of the excitation light.

10. The method of claim 1, further comprising:

comparing the fluorescence intensity with a database of fluorescence intensities of known sugar chains; and

determining the identity of the labeled sugar chain by selecting a sugar chain of known structure having a matching pattern of fluorescence intensity.

5. A glass substrate coated with a compound comprising an epoxy group as an active group, onto which a protein that interacts with a sugar chain has been immobilized, and in which a number of reaction vessels have been formed by affixing a rubber having a number of holes onto the glass.

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

7. The substrate of claim 5, 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.

13. The glass substrate of claim 5, wherein the rubber is a black silicon rubber.

11. A method for analyzing an interaction between a sugar chain and a protein that interacts with a sugar chain, comprising:

contacting a sample comprising at least one fluorescently labeled glycoprotein with a glass slide comprising one or more lectin conjugated to the glass slide through an epoxy group of 3-glycidoxypropyl trimethoxysilane;

applying an excitation light to the substrate from the edge of the glass substrate without washing the glass substrate;

generating an evanescent wave by total internal reflection of the excitation light; and

measuring intensity of emitted fluorescent light generated by the evanescent wave, wherein an increase in the emitted fluorescent light indicates the interaction between the fluorescently labeled glycoprotein and the lectin.

12. The method of claim 11, wherein the rubber is a black silicon rubber.