ANALYZER FOR GLYCAN OR COMPLEX CARBOHYDRATE

• US 20120053089A1
• Filed: 08/19/2011
• Published: 03/01/2012
• Est. Priority Date: 06/23/2005
• 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, Kuno, Atsushi, Uchiyama, Noboru
• Granted Patent: US 8,597,576 B2
• Time in Patent Office: Days
• US Class Current: 506/18
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• 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: G01N 33/548 Carbohydrates, e.g. dextran

Abstract

An analyzer of the present invention for analyzing a glycan or a complex glycan includes a substrate, a fluorescent labeling excitation means and a fluorescent intensity measurement means.

The substrate comprises a rectangle photoconductive base plate coated by a compound containing an active group to fix protein by an amino group thereof, one and more open-topped reaction vessel formed on a surface of the base plate, and plural spots of glycan binding proteins arranged in a matrix and immobilized on the surface of the base plate in the reaction vessel.

The fluorescent labeling excitation means which excites fluorescent label by generating evanescent-waves on the surface of the substrate comprises a pair of optical fibers arranged toward the both side end faces of the right and left of the substrate to introduce a light thereinto.

The fluorescent intensity measurement means for measuring the intensity of fluorescence generated by the fluorescent labeling excitation means on every spots of arranging the glycan binding protein.

By using this analyzer, a glycan or a complex carbohydrate can be analyzed conveniently, quickly, sensitively and accurately.

Claims

1. An analyzer for fluorescence-labeling a glycan to be detected or a complex carbohydrate to be detected with or without purification, measuring the binding state of the glycan or the complex carbohydrate and a protein thereby, and analyzing the glycan to be detected or the complex carbohydrate to be detected, or a specimen containing the same,

including

a substrate comprising

a rectangle photoconductive base plate coated by a compound containing an active group to fix protein by an amino group thereof,

one and more open-topped reaction vessel formed on a surface of the base plate, and

plural spots of glycan binding proteins arranged in a matrix and immobilized on the surface of the base plate in the reaction vessel;

a fluorescent labeling excitation means which excites fluorescent label by generating evanescent-waves on the surface of the substrate comprises a pair of optical fibers arranged toward the both side end faces of the right and left of the substrate to introduce a light thereinto; and

a fluorescent intensity measurement means for measuring the intensity of fluorescence generated by the fluorescent labeling excitation means on every spots of arranging the glycan binding protein.

2. An analyzer according to claim 1, wherein

the surface of the photoconductive base plate is covered by a light blocking rubber opening holes at the positions corresponding to areas of the reaction vessels so that the reaction vessels are formed in areas of the holes.

3. An analyzer according to claim 1, wherein

the surface of the photoconductive base plate is coated by a water-repellent material except for the areas corresponding to the reaction vessels so that the reaction vessels are formed in areas surrounded by the water-repellent material.

4. An analyzer according to claim 1, wherein

the glycan binding protein is a lectin or a glycan recognition antibody belonging to an IgM class.

5. An analyzer according to claim 1, wherein

the substrate is one in which the glycan binding proteins are arranged and immobilized in a predetermined pattern in accordance with the type thereof.

6. An analyzer according to claim 1, wherein

an antibody to a portion other than the glycan of the complex carbohydrate are arranged and immobilized together with the glycan binding protein on the substrate.

7. An analyzer according to claim 1, having a memory means for storing the fluorescent intensity measured by the fluorescent intensity measurement means on every position of arranging the glycan binding proteins corresponding to the glycan to be detected or the complex carbohydrate to be detected that are used.

8. An analyzer according to claim 1, having a memory means for storing the measured fluorescent intensity divided by steps on every position of arranging the glycan binding protein.

9. An analyzer according to claim 8, having an indication means for indicating the fluorescent intensity divided stepwise on every type of each of the glycan binding proteins.

10. An analyzer according to claim 1, wherein

the substrate is one in which multiple types of identical glycan binding proteins are arranged and immobilized on every type thereof.

11. An analyzer according to claim 10, having a calculation and memory means for calculating and storing an average value of the fluorescent intensity measured for identical glycan binding proteins.

12. An analyzer according to claim 11, having a memory means for storing stepwise average values of fluorescent intensities measured for identical glycan binding proteins.

13. An analyzer according to claim 12, having an indication means for indicating stepwise the fluorescent intensities being divided on every type of the glycan binding proteins.

14. An analyzer according to claim 7, wherein

the fluorescent intensity information for known glycans or complex carbohydrates are stored in the memory means.

15. An analyzer according to claim 8 wherein

the fluorescent intensity information for known glycans or complex carbohydrates are stored in the memory means.

16. An analyzer according to claim 11, wherein

the fluorescent intensity information for known glycans or complex carbohydrates are stored in the memory means.

17. An analyzer according to claim 12, wherein

the fluorescent intensity information for known glycans or complex carbohydrates are stored in the memory means.

18. An analyzer according to claim 14, having a selection means for matching the fluorescent intensity information for a glycan or a glycoside protein as a target of analysis to the fluorescent intensity information for known glycans or complex carbohydrates, and selecting an identical or approximate glycan or complex carbohydrate.

19. An analyzer according to claim 15, having a selection means for matching the fluorescent intensity information for a glycan or a glycoside protein as a target of analysis to the fluorescent intensity information for known glycans or complex carbohydrates, and selecting an identical or approximate glycan or complex carbohydrate.

20. An analyzer according to claim 16, having a selection means for matching the fluorescent intensity information for a glycan or a glycoside protein as a target of analysis to the fluorescent intensity information for known glycans or complex carbohydrates, and selecting an identical or approximate glycan or complex carbohydrate.

21. An analyzer according to claim 17, having a selection means for matching the fluorescent intensity information for a glycan or a glycoside protein as a target of analysis to the fluorescent intensity information for known glycans or complex carbohydrates, and selecting an identical or approximate glycan or complex carbohydrate.