Immunoassay element

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.

What is claimed is:  
     
         1 . An immunoassay element for quantitatively analyzing an antigen by determining the change in enzymatic activity caused by any of 
 1) 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.