Enzyme-labeled immunoassay and device therefor

Abstract

An enzyme-labeled immunoassay is performed by the steps of allowing a test sample to react with an enzyme-labeled reagent, allowing a substrate to react with the enzyme to form a signal, and immobilising the enzyme-labeled reagent, with the prevention of a further signal formation from a predetermined time on after the immobilisation of the enzyme-labeled reagent, using an enzyme inhibitor. A device for performing this enzyme-labeled immunoassay includes an absorbent material capable of transporting a developing solution by capillary action, the absorbent material including (a) a developing liquid application zone, (b) an enzyme-labeled reagent zone containing an enzyme-labeled reagent, (c) a sample receiving zone, and (d) an indicator reagent zone capable of immobilising the enzyme-labeled reagent after the reaction of the test sample with the enzyme-labeled reagent in an amount dependent on the assay result, with an enzyme inhibitor being applied to a portion in the absorbent material upstream of the enzyme-labeled reagent zone, which enzyme inhibitor prevents the formation of a signal from a predetermined time on after the enzyme-labeled reagent is immobilised at the indicator reagent zone.

What is claimed is:  
     
         1 . An enzyme-labeled immunoassay comprising the steps of: 
 allowing a test sample to react with an enzyme-labeled reagent,    allowing a substrate to react with said enzyme to form a signal, and    immobilising said enzyme-labeled reagent, with the prevention of a further signal formation from a predetermined time on after the immobilisation of said enzyme-labeled reagent, using an enzyme inhibitor.    
     
     
         2 . The enzyme-labeled immunoassay as claimed in    claim 1   , wherein said enzyme is phosphatase, and said enzyme inhibitor is selected from the group consisting of a phosphoric acid compound and a chelate compound.  
     
     
         3 . The enzyme-labeled immunoassay as claimed in    claim 2   , wherein said phosphoric acid compound is selected from the group consisting of phosphoric acid, phosphate, phosphoric monoester, naphtholphosphoric acid, glycerophosphoric acid, phenylphosphate, phosphoethanol-amine, phosphorylcholin, and glycose phosphoric acid.  
     
     
         4 . The enzyme-labeled immunoassay as claimed in    claim 2   , wherein said chelate compound is selected from the group consisting of ethylenediaminetetraacetic acid, phenanthroline, and ethyleneglycosetetraacetic acid.  
     
     
         5 . The enzyme-labeled immunoassay as claimed in    claim 1   , wherein said enzyme is peroxidase, and said enzyme inhibitor is selected from the group consisting of a reducing compound and an azide compound.  
     
     
         6 . The enzyme-labeled immunoassay as claimed in    claim 1   , wherein said enzyme is β-galactosidase, and said enzyme inhibitor is galactose.  
     
     
         7 . A device for performing an enzyme-labeled immunoassay comprising an absorbent material capable of transporting a developing solution by capillary action, said absorbent material comprising (a) a developing liquid application zone to which said developing liquid is applied, (b) an enzyme-labeled reagent zone comprising an enzyme-labeled reagent, (c) a sample receiving zone to which a test sample is applied, and (d) an indicator reagent zone comprising an indicator reagent capable of immobilising said enzyme-labeled reagent after the reaction of said test sample with said enzyme-labeled reagent in an amount dependent on the assay result, which zones are sequentially arranged in the direction of the transport of said developing solution, with a substrate for an enzyme being applied to a portion in said absorbent material upstream of said enzyme-labeled reagent zone, and an enzyme inhibitor being applied to a portion in said absorbent material upstream of said enzyme-labeled reagent zone, which enzyme inhibitor prevents a signal formation which occurs by the reaction of said enzyme and said substrate, from a predetermined time on after said enzyme-labeled reagent is immobilised at said indicator reagent zone.  
     
     
         8 . The device as claimed in    claim 7   , wherein said developing solution further comprises said enzyme inhibitor.  
     
     
         9 . The device as claimed in    claim 7   , further comprising an enzyme inhibitor zone comprising said enzyme inhibitor, which is disposed upstream of said enzyme-labeled reagent zone.  
     
     
         10 . The device as claimed in    claim 7   , wherein said enzyme is phosphatase, and said enzyme inhibitor is selected from the group consisting of a phosphoric acid compound and a chelate compound.  
     
     
         11 . The device as claimed in    claim 10   , wherein said phosphoric acid compound is selected from the group consisting of phosphoric acid, phosphate, phosphoric monoester, naphtholphosphoric acid, glycerophosphoric acid, phenylphosphate, phosphoethanol-amine, phosphorylcholin, and glycose phosphoric acid.  
     
     
         12 . The device as claimed in    claim 10   , wherein said chelate compound is selected from the group consisting of ethylenediaminetetraacetic acid, phenanthroline, and ethyleneglycosetetraacetic acid.  
     
     
         13 . The device as claimed in    claim 7   , wherein said enzyme is peroxidase, and said enzyme inhibitor is selected from the group consisting of a reducing compound and an azide compound.  
     
     
         14 . The device as claimed in    claim 7   , wherein said enzyme is β-galactosidase, and said enzyme inhibitor is galactose.