Enzyme-labeled immunoassay and device therefor

• US 20010005585A1
• Filed: 02/08/2001
• Published: 06/28/2001
• Est. Priority Date: 04/23/1997
• Status: Abandoned Application

• Current Assignee: Fujirebio Inc. (H.U. Group Holdings, Inc.)
• Original Assignee: Fujirebio Inc. (H.U. Group Holdings, Inc.)
• Inventors: Isomura, Mitsuo, Ashihara, Yoshihiro, Sato, Atsuka
• Application Number:
  US09/778,864
• Publication Number:
  US 20010005585A1
• Time in Patent Office: Days
• US Class Current: 435/7.95
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

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.

Claims

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.