Immunoassay element

• US 20050084953A1
• Filed: 10/07/2004
• Published: 04/21/2005
• Est. Priority Date: 06/13/1997
• Status: Abandoned Application

• Current Assignee: Fuji Photo Film Co Limited (Fujifilm Holdings Corporation)
• Original Assignee: Fuji Photo Film Co Limited (Fujifilm Holdings Corporation)
• Inventors: Mori, Toshihiro, Ito, Hitomi, Amano, Yoshikazu, Sakaino, Yoshiki, Seshimoto, Osamu, Ito, Toshihisa
• Application Number:
  US10/961,459
• Publication Number:
  US 20050084953A1
• Time in Patent Office: Days
• US Class Current: 435/287.200
• CPC Class Codes: G01N 2333/924 acting on glycosyl compounds (3.2)
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: G01N 33/54391 based on vertical flow
• CPC Class Codes: Y10S 435/966 involving an enzyme system with high turnover rate or complement magnified assay, e.g. multi-enzyme systems
• CPC Class Codes: Y10S 435/969 Multiple layering of reactants
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

The immunoassay element for quantitatively analyzing an antigen by determining the change in enzymatic activity of an enzyme-labelled antigen or antibody caused by an immunological reaction. The immunoassay element comprises a substrate layer containing a non-diffusible substrate which forms a diffusible material in the presence of the labelling enzyme, and a reagent layer containing a fragmenting enzyme for further fragmenting the diffusible material into a lower molecular weight product. As the non-diffusible substrate, a substrate capable of reacting solely with the lebelling enzyme and incapable of reacting the fragmenting enzyme is utilized. When an endo-active glucosidase is used as the labelling enzyme, and an exo-active glucosidase is used the fragmenting enzyme in the reagent layer, the non-diffusible substrate of the substrate layer is preferred to be an endo type selectively reactive substrate, which means a substrate having a reactivity specific to endo-active glucosidase. Highly sensitive assay is realized with high accuracy and high reproducibility and good storage stability.

Claims

1. An immunoassay element for quantitatively analyzing an antigen by determining the change in enzymatic activity caused by any of a reaction between the antigen and an enzyme-labelled antibody;

2) a reaction between the antigen, an antibody and an enzyme-labelled antigen; and

3) a reaction between the antigen, an enzyme-labelled antibody and a conjugate of the antigen with a high molecular weight compound;

wherein said element comprises a substrate layer containing a non-diffusible substrate which forms a diffusible material in the presence of said labelling enzyme, and a reagent layer containing a fragmenting enzyme for further fragmenting said diffusible material into a lower molecular weight product;

characterized in that said non-diffusible substrate is a substrate which reacts solely with said labelling enzyme and avoids reacting with said fragmenting enzyme.

2. The immunoassay element according to claim 1, wherein said non-diffusible substrate is a macromolecular polysaccharide, said labelling enzyme is an endo-active glucosidase, and said fragmenting enzyme is an exo-active glucosidase.

3. The immunoassay element according to claim 1, wherein said labelling enzyme is an endo-active glucosidase, said fragmenting enzyme is an exo-active glucosidase, and said non-diffusible substrate is an endo-type selectively reactive substrate having a modified non-reducing terminal glucose.

4. The immunoassay element according to claim 3, wherein said endo-type selectively reactive substrate has the non-reducing terminal glucose thereof modified with a carboxylmethyl group.

5. The immunoassay element according to claim 3, wherein said labelling enzyme is α

-amylase, said fragmenting enzyme is glucoamylase or α

-glucosidase, and said non-diffusible substrate is carboxylmethylated starch restrictively decomposed in advance with an endo-active glucosidase from the non-reducing terminal glucose site through the carboxylmethyl-modified glucose unit site or the glucose chain branching site.

6. The immunoassay element according to claim 1, wherein said reagent layer is formed of a layer containing a hydrophilic polymer as the binder.

7. The immunoassay element according to claim 1, wherein said substrate layer is a porous layer formed of a porous medium.

8. The immunoassay element according to claim 1, wherein said enzyme-labelled antibody is contained in said substrate layer or a layer superposed on said substrate layer.

9. The immunoassay element according to claim 1, wherein said antibody is contained in said substrate layer or a layer superposed on said substrate layer.

10. The immunoassay element according to claim 1, wherein said enzyme-labelled antigen is contained in said substrate layer or a layer superposed on said substrate layer.

11. The immunoassay element according to claim 1, wherein said antibody and said enzyme-labelled antigen are contained in said substrate layer or a layer superposed on said substrate layer.

12. The immunoassay element according to claim 1, wherein said conjugate of the antigen with the high molecular weight compound and said enzyme-labelled antibody are contained in said substrate layer or a layer superposed on said substrate layer.

13. The immunoassay element according to claim 1, further comprising a reagent composition for reacting with said lower molecular weight product to form a dye having an absorption peak in the visible wavelength range and contained in said reagent layer or another water-permeable layer.

14. The immunoassay element according to claim 13, wherein the reagent composition contains a leuco dye which colors upon oxidation.

15. The immunoassay element according to claim 14, wherein said reagent layer contains a hydrophilic binder, and the reagent composition contains a dispersion of a solution of leuco dye in a water-insoluble solvent in the hydrophilic binder.

16. The immunoassay element according to claim 15, wherein said reagent composition contains a peroxidase and the leuco dye.

17. An immunoassay element for quantitatively analyzing an antibody by determining the change in enzymatic activity caused by a reaction between the antibody and an enzyme-labelled antigen or a reaction between the antibody, an antigen and an enzyme-labelled antibody, wherein said element comprises a substrate layer containing a non-diffusible substrate which forms a diffusible material in the presence of the labelling enzyme, and a reagent layer containing a fragmenting enzyme for further fragmenting said diffusible material into a lower molecular weight product:

characterized in that said non-diffusible substrate is a substrate which reacts solely with said labelling enzyme and avoids reacting with said fragmenting enzyme.

18. The immunoassay element according to claim 17, wherein said non-diffusible substrate is a macromolecular polysaccharide, said labelling enzyme is an endo-active glucosidase, and said fragmenting enzyme is an exo-active glucosidase.

19. The immunoassay element according to claim 17, wherein said labelling enzyme is an endo-active glucosidase, said fragmenting enzyme is an exo-active glucosidase, and said non-diffusible substrate is an endo-type selectively reactive substrate having a modified non-reducing terminal glucose.

20. The immunoassay element according to claim 19, wherein said endo-type selectively reactive substrate has the non-reducing terminal glucose thereof modified with a carboxylmethyl group.

21. The immunoassay element according to claim 19, wherein said labelling enzyme is α

-amylase, said fragmenting enzyme is glucoamylase or α

-glucosidase, and said non-diffusible substrate is carboxylmethylated starch restrictively decomposed in advance with an endo-active glucosidase from the non-reducing terminal glucose site through the carboxylmethyl-modified glucose unit site or the glucose chain branching site.

22. The immunoassay element according to claim 17, wherein said reagent layer is formed of a layer containing a hydrophilic polymer as the binder.

23. The immunoassay element according to claim 17, wherein said substrate layer is a porous layer formed of a porous medium.

24. The immunoassay element according to claim 17, wherein said enzyme-labelled antibody is contained in said substrate layer or a layer superposed on said substrate layer.

25. The immunoassay element according to claim 17, wherein said antibody is contained in said substrate layer or a layer superposed on said substrate layer.

26. The immunoassay element according to claim 17, wherein said enzyme marker antigen is contained in said substrate layer or a layer superposed on said substrate layer.

27. The immunoassay element according to claim 17, wherein said antibody and said enzyme-labelled antigen are contained in said substrate layer or a layer superposed on said substrate layer.

28. The immunoassay element according to claim 17, further comprising a reagent composition for reacting with said lower molecular weight product to form a dye having an absorption peak in the visible wavelength range and contained in said reagent layer or another water-permeable layer.

29. The immunoassay element according to claim 28, wherein the reagent composition contains a leuco dye which colors upon oxidation.

30. The immunoassay element according to claim 29, wherein said reagent layer contains a hydrophilic binder, and the reagent composition contains a dispersion of a solution of leuco dye in a water-insoluble solvent in the hydrophilic binder.

31. The immunoassay element according to claim 30, wherein said reagent composition contains a peroxidase and the leuco dye.