Immunoassay element

• US 7,820,402 B2
• Filed: 09/19/2005
• Issued: 10/26/2010
• Est. Priority Date: 06/13/1997
• Status: Expired due to Fees

• 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, Ito, Toshihisa, Seshimoto, Osamu
• Primary Examiner(s): Cheu; Jacob
• Assistant Examiner(s): Do; Pensee T
• Application Number:
  US11/230,127
• Publication Number:
  US 20060024770A1
• Time in Patent Office: 1,863 Days
• Field of Search: 435/7.9, 435/7.91, 435/7.92, 435/14, 435/18, 435/22, 435/28, 435/25, 435/966, 435/969, 435/970, 436/518, 436/528, 436/531, 436/537, 436/807, 436/810, 422/56, 422/57
• US Class Current: 435/7.9
• 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 labelling 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 a change in enzymatic activity caused by any of followings:

1) a reaction between the antigen and an enzyme-labelled antibody, the enzyme-labelled antibody being a conjugate of an antibody and a labelling enzyme, the antibody being capable to react and bind specifically with the antigen, wherein the antigen sterically hinders enzymatic activity in a specific binding complex of the antigen and the enzyme-labelled antibody;

2) a reaction between the antigen, an antibody and an enzyme-labelled antigen, the enzyme-labelled antigen being a conjugate of an antigen and a labelling enzyme, wherein the antibody sterically hinders enzymatic activity in a specific binding complex of the antibody and the 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, the enzyme-labelled antibody being a conjugate of an antibody and a labelling enzyme, wherein the conjugate of the antigen with a high molecular weight compound sterically hinders enzymatic activity in a specific binding complex of the conjugate of the antigen with a high molecular weight compound and the enzyme-labelled antibody;

said element comprising;

a substrate layer containing a non-diffusible substrate composed of polysaccharide which is capable of being fragmented by said labelling enzyme into a diffusible glucose oligomer which migrates from said substrate layer; and

a reagent layer containing a fragmenting enzyme for further fragmenting said diffusible glucose oligomer, which has migrated from said substrate layer, into a detectable glucose monomer, said substrate layer being superposed on said reagent layer;

wherein said labelling enzyme is α

-amylase, and said fragmenting enzyme is glucoamylase or α

-glucosidase; and

wherein said non-diffusible substrate is carboxylmethylated starch restrictively decomposed in advance with an exo-active glucosidase from the non-reducing terminal glucose site through the carboxylmethyl-modified glucose unit site or the glucose chain branching site so that said non-diffusible substrate reacts solely with said labelling enzyme and avoids reacting with said fragmenting enzyme.

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

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

4. 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.

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

6. 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.

7. 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.

8. 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.

9. The immunoassay element according to claim 1, further comprising a reagent composition for reacting with said detectable glucose monomer to form a dye having an absorption peak in the visible wavelength range and contained in said reagent layer or another water permeable layer.

10. The immunoassay element according to claim 9, wherein the reagent composition contains a leuco dye which colors upon oxidation.

11. The immunoassay element according to claim 10, 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.

12. The immunoassay element according to claim 11, wherein said reagent composition contains a peroxidase and the leuco dye.

13. An immunoassay element for quantitatively analyzing an antibody by determining a change in enzymatic activity caused by any of followings:

a reaction between the antibody and an enzyme-labelled antigen, the enzyme-labelled antigen being a conjugate of an antigen and a labelling enzyme, the antibody being capable to react and bind specifically with the antigen, wherein the antibody sterically hinders enzymatic activity in a specific binding complex of the antibody and the enzyme-labelled antigen; and

a competitive reaction between the antibody, an antigen and an enzyme-labelled antibody, the enzyme-labelled antibody being a conjugate of an antibody and a labelling enzyme, wherein the antigen sterically hinders enzymatic activity in a specific binding complex of the antigen and the enzyme-labelled antibody;

said element comprising;

a substrate layer containing a non-diffusible substrate composed of polysaccharide which is capable of being fragmented by said labelling enzyme into a diffusible glucose oligomer which migrates from said substrate layer; and

a reagent layer containing a fragmenting enzyme for further fragmenting said diffusible glucose oligomer, which has migrated from said substrate layer, into a detectable glucose monomer, said substrate layer being superposed on said reagent layer;

wherein said labelling enzyme is α

-amylase, and said fragmenting enzyme is glucoamylase or α

-glucosidase; and

wherein said non-diffusible substrate is carboxylmethylated starch restrictively decomposed in advance with an exo-active glucosidase from the non-reducing terminal glucose site through the carboxylmethyl-modified glucose unit site or the glucose chain branching site so that said non-diffusible substrate reacts solely with said labelling enzyme and avoids reacting with said fragmenting enzyme.

14. The immunoassay element according to claim 13, wherein said reagent layer is formed of a layer containing a hydrophilic polymer as the binder.

15. The immunoassay element according to claim 13, wherein said substrate layer is a porous layer formed of a porous medium.

16. The immunoassay element according to claim 13, wherein said enzyme labelled antibody is contained in said substrate layer or a layer superposed on said substrate layer.

17. The immunoassay element according to claim 13, wherein said antibody is contained in said substrate layer or a layer superposed on said substrate layer.

18. The immunoassay element according to claim 13, wherein said enzyme-labelled antigen is contained in said substrate layer or a layer superposed on said substrate layer.

19. The immunoassay element according to claim 13, wherein said antibody and said enzyme labelled antigen are contained in said substrate layer or a layer superposed on said substrate layer.

20. The immunoassay element according to claim 13, further comprising a reagent composition for reacting with said detectable glucose monomer to form a dye having an absorption peak in the visible wavelength range and contained in said reagent layer or another water permeable layer.

21. The immunoassay element according to claim 20, wherein the reagent composition contains a leuco dye which colors upon oxidation.

22. The immunoassay element according to claim 21, 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.

23. The immunoassay element according to claim 22, wherein said reagent composition contains a peroxidase and the leuco dye.