Analyzer for glycan or complex carbohydrate

• US 8,597,576 B2
• Filed: 08/19/2011
• Issued: 12/03/2013
• 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
• Primary Examiner(s): MERKLING, SALLY ANNE
• Application Number:
  US13/213,596
• Publication Number:
  US 20120053089A1
• Time in Patent Office: 837 Days
• Field of Search: 435/7.1, 435/6.16, 435/325, 422/400, 422/82.05
• US Class Current: 422/82.05
• 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.

Claims

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

plural open-topped reaction vessels arranged in a matrix with plural columns in a direction along a short side of the base plate and 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 exciter, which excites fluorescent label by generating evanescent-waves on the surface of the substrate, comprising a pair of optical fibers arranged toward both right and left side end faces as long sides of the substrate to introduce a light symmetrically in a direction along the short side thereinto; and

a fluorescent intensity measurer for measuring the intensity of fluorescence generated by the fluorescent labeling exciter on each of the spots of the glycan binding protein.

2. The 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. The 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. The analyzer according to claim 1, wherein the glycan binding protein is a lectin or a glycan recognition antibody belonging to an IgM class.

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

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

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

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

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

14. The analyzer according to claim 7, wherein the fluorescent intensity information for known glycans or complex carbohydrates are stored in the memory.

15. The analyzer according to claim 8 wherein the fluorescent intensity information for known glycans or complex carbohydrates are stored in the memory.

16. The analyzer according to claim 11, wherein the fluorescent intensity information for known glycans or complex carbohydrates are stored in the memory.

17. The analyzer according to claim 12, wherein the fluorescent intensity information for known glycans or complex carbohydrates are stored in the memory.

18. The analyzer according to claim 14, having a selector 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. The analyzer according to claim 15, having a selector 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. The analyzer according to claim 16, having a selector 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. The analyzer according to claim 17, having a selector 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.