Multizone analytical element having detectable signal concentrating zone

• US 4,806,312 A
• Filed: 08/28/1985
• Issued: 02/21/1989
• Est. Priority Date: 08/28/1985
• Status: Expired due to Term

• Current Assignee: Miles, Inc. (Bayer AG)
• Original Assignee: Miles, Inc. (Bayer AG)
• Inventors: Greenquist, Alfred C.
• Primary Examiner(s): Castel, Benoit
• Assistant Examiner(s): Johnston, Jill
• Application Number:
  US06/770,237
• Time in Patent Office: 1,273 Days
• Field of Search: 436/807, 436/810, 436/815, 422/50, 422/57, 422/58, 435/7, 435/805
• US Class Current: 422/424
• CPC Class Codes: G01N 33/525 Multi-layer analytical elements
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• 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
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds

Abstract

A multizone test device for the determination of analyte from a liquid test medium upon contact with the liquid test medium and a labeled reagent comprising a chemical group having a detectable chemical property. The test device preferably comprises multilayers including a reagent layer incorporated with an immobilized reagent and a detection layer incorporated with an immobilized form of an interactive detection reagent for the labeled reagent. The immobilized reagent in the reagent layer and the labeled reagent comprise specific binding partners which will bind to each other dependent upon the amount of analyte present. Labeled reagent which does not become bound to the immobilized reagent in the reagent layer migrates into the detection layer and interacts with the immobilized interactive detection reagent therein which results in the localized generation of a detectable reaction product which preferably is also immobilized in the detection zone. As a result, reverse migration of the labeled reagent, and preferably the detectable reaction product from the detection layer is prevented and the detectable chemical property provided by the label of the labeled reagent is localized in the detection layer for the precise measurement thereof and correlation to the amount of analyte in the test medium.

Claims

1. A multizone test device for the specific binding assay determination of an analyte in a liquid test medium involving binding between (i) the analyte and a labeled or immobilized form of the analyte or of a binding analog thereof, and (ii) an immobilized or labeled form, respectively, of a binding partner of the analyte, the labeled one of the analyte, analog thereof, or binding partner being a labeled reagent comprising a detectable chemical group which interacts with a detectant composition to produce a detectable signal, the test device comprising, in fluid flow contact,

(1) a reagent zone comprising a solid, porous matrix incorporated with a immobilized one of the analyte, analog thereof, or binding partner, and

(2) a detection zone comprising a solid, porous matrix for receiving and measuring labeled reagent which migrates into such zone and incorporated with an immobilized form of at least one component of a detectant composition.

2. The test device of claim 1 wherein the detectant component is immobilized in the detection zone by being covalently coupled to the matrix comprised therein.

3. The test device of claim 1 wherein the detectant component is immobilized in the detection zone by being attached to a high molecular weight polymeric substance dispersed in said matrix.

4. The test device of claim 1 wherein a binding partner of the analyte is incorporated in said reagent zone and is an antibody or a fragment thereof.

5. The test device of claim 1 which comprises a solid, porous chromatographic element and wherein the reagent and detection zones are discrete sections of such element.

6. The test device of claim 1 wherein the labeled reagent is incorporated in said reagent zone and the detectable chemical group in the labeled reagent comprises a fluorescer and wherein the detectant component immobilized in the detention zone is a quencher for the fluorescence of the labeled reagent.

7. The test device of claim 6 wherein the detectable chemical group in the labeled reagent is a polymer residue bearing multiple fluorescers.

8. The test device of claim 1 wherein the reagent and detection zones are in the form of layers in fluid flow contact.

9. The test device of claim 8 which additionally comprises a reagent layer comprising a solid, porous matrix incorporated with a test medium soluble form of the labeled reagent.

10. The test device of claim 8 which additionally comprises a support element situated on the opposite side of the detection layer from the reagent layer.

11. The test device of claim 1 wherein the detectant component immobilized in the detection zone participates in a chemical reaction with the labeled reagent to produce a product which provides a detectable signal.

12. The test device of claim 11 wherein the immobilized detectant component undergoes a chemical reaction with the labelec reagent to form an immobilized product which provides the detectable signal.

13. The test device of claim 11 wherein the detectable product is insoluble.

14. The test device of claim 11 wherein the detectable product is soluble and wherein the detection zone additionally comprises an immobilizing agent for said detectable product.

15. The test device of claim 11 wherein the labeled reagent is incorporated in said reagent zone and detectable chemical group in the labeled reagent is an enzymatically active group.

16. The test device of claim 15 wherein the detectable chemical group in the labeled reagent is (i) a enzyme or (ii) a substrate or cofactor for such enzyme, and wherein the immobilized detectant component is the other thereof.

17. The test device of claim 4 wherein the detectable chemical group in the labeled reagent is a polymer residue to which are attached internally-quenched multiple fluorescers and wherein the immobilized detectant component is an enzyme which acts on the fluorescer-polymer residue to cleave and release unquenched fluorescer molecules.

18. The test device of claim 16 wherein the detectable chemical group in the labeled reagent is an enzyme and wherein the immobilized detectant component is a substrate therefor.

19. The test device of claim 18 wherein the substrate is chromogenic.

20. The test device of claim 18 wherein the substrate is fluorogenic.

21. The test device of claim 18 wherein the substrate is chemiluminescent.

22. A multilayer immunoassay test device for the determination of an analyte in an aqueous liquid medium, which test device provides a detectable optical signal upon contact with aqueous medium containing analyte, the test device comprising, in fluid flow contact and in the following ordered sequence,

(1) a first reagent layer comprising a solid, porous matrix incorporated with a water soluble form of a labeled reagent comprising an antibody, or a fragment thereof, for the analyte and an enzyme that acts on an enzyme substrate to transform it into a procuct which provides the detectable optical signal,

(2) a second reagent layer comprising a solid, porous matrix incorporated with an immobilized form of the analyte or a binding analog thereof,

(3) a detection layer comprising a solid, porous matrix for receiving and measuring labeled reagent which migrates into such layer and incorporated with an immobilized form of said enzyme substrate, and

(4) a support element comprising a solid, nonporous substrate.

23. The test device of claim 22 wherein the substrate is chromogenic.

24. The test device of claim 22 wherein the substrate is fluorogenic.

25. The test device of claim 22 wherein the substrate is chemiluminescent.

26. The test device of claim 22 wherein the antibody fragment is derived from a monoclonal antibody.

27. The test device of claim 22 wherein the detectant component is immobilized in the detection zone by being covalently coupled to the matrix comprised therein.

28. The test device of claim 22 wherein the detectant component is immobilized in the detection zone by being attached to a high molecular weight polymeric substance dispersed in said matrix.

29. The test device of claim 22 wherein said first reagent layer, second reagent layer, and detection layer are transparent to the detectable optical signal, and said support element is opaque to such signal.