Reversible immobilization of assay reagents in a multizone test device

• US 4,959,305 A
• Filed: 10/31/1988
• Issued: 09/25/1990
• Est. Priority Date: 06/18/1986
• Status: Expired due to Fees

• Current Assignee: Miles, Inc. (Bayer AG)
• Original Assignee: Miles, Inc. (Bayer AG)
• Inventors: Woodrum, David L.
• Primary Examiner(s): Warden, Robert J.
• Application Number:
  US07/265,303
• Time in Patent Office: 694 Days
• Field of Search: 422/56-58, 435/7, 435/805, 436/170
• US Class Current: 435/7.7
• CPC Class Codes: G01N 33/525 Multi-layer analytical elements
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/97 Test strip or test slide

Abstract

A multizone test device for the determination of analyte in a liquid test medium. The test device preferably comprises multilayers including a first layer comprising a solid, porous matrix incorporated with a reversibly immobilized first reagent and a second layer comprising a solid, porous matrix incorporated with a second reagent which interacts with the first reagent and analyte from the liquid test medium to provide a detectable signal. A reversible binding interaction between the first reagent and the matrix of the first layer prevents the first reagent from prematurely migrating into the second layer during manufacture of the device and prior to application of the liquid test medium to the test device. The reversible binding interaction is sufficiently disruptable by contact of the first layer with the liquid test medium to release and thereby render diffusible an analytically effective amount of the first reagent within the test device.

Claims

1. A multizone test device for the immunoassay determination of an antigen or hapten analyte in a liquid test medium, comprising, in fluid flow contact,

(a) a reagent zone comprising a solid, porous matrix and a labeled reagent reversibly immobilized therein by a binding interaction between the labeled reagent and the matrix that is sufficiently disruptable by contact of the matrix with a predetermined component of the test medium other than the analyte to release and thereby render diffusible an analytically effective amount of the labeled reagent independent of the presence or amount of analyte in the liquid test medium, the labeled reagent comprising one of the pair

(i) the analyte or a binding analog thereof, and

(ii) an antibody which binds the analyte labeled with a detectable chemical group,

(b) a reaction zone comprising a solid, porous matrix incorporated with an immobilized form of the other of said pair which interacts with both of the analyte and the labeled reagent to bind and thereby immobilize labeled reagent while leaving an amount of the labeled reagent diffusible as a function of the amount of analyte in the liquid test medium, and

(c) a detection zone comprising a solid, porous matrix for receiving labeled reagent which is free to diffuse thereinto from the reaction zone to provide a detectable signal related to the amount of analyte present in the liquid test medium.

2. The test device of claim 1 wherein the reversible binding interaction is an anionic binding interaction between the labeled reagent and the reagent zone matrix and wherein the liquid test medium comprises a salt solution.

3. The test device of claim 2 wherein the reagent layer matrix comprises an ion exchange material.

4. The test device of claim 3 wherein the ion exchange material is an anion exchange material selected from the group consisting of diethylaminoethyl cellulose, epichlorohydrin triethanolamine cellulose, triethylaminoethyl cellulose bromide and polyethyleneimine.

5. The test device of claim 3 wherein the ion exchange material is a cation exchange material selected from the group consisting of carboxymethyl cellulose, phosphate cellulose and dextran sulfate.

6. The test device of claim 1 wherein the reversible binding interaction is a reversible covalent binding interaction between the labeled reagent and the reagent zone matrix and wherein the liquid test medium comprises a reducing agent.

7. The test device of claim 1 wherein the reversible binding interaction is a hydrophobic binding interaction between the labeled reagent and the reagent zone matrix and wherein the liquid test medium comprises a non-ionic detergent.

8. The test device of claim 1 wherein the reversible binding interaction is a reversible biochemical binding affinity between the labeled reagent and the reagent zone matrix and wherein the liquid test medium comprises a competing ligand.

9. The test device of claim 1 wherein the liquid test medium comprises an undiluted biological fluid test sample capable of disrupting the binding interaction between the reagent zone matrix and the labeled reagent.

10. The test device of claim 1 wherein the liquid test medium comprises a mixture of a biological fluid test sample and a diluent capable of disrupting the binding interaction between the reagent zone matrix and the first reagent.

11. The test device of claim 1 wherein the biological liquid test medium is whole blood, blood serum, blood plasma, amniotic fluid, cerebrospinal fluid or urine.

12. The test device of claim 1 wherein the detectable chemical group of the labeled reagent is coupled to the analyte, binding analog, or antibody by a linking group.

13. The test device of claim 12 wherein the reversible binding interaction is between the matrix of the reagent zone and the detectable chemical group or the linking group of the labeled reagent.

14. The test device of claim 11 wherein the detectable chemical group in the labeled reagent possesses a physical property which is detectable in the detection zone.

15. The test device of claim 14 wherein the detectable chemical group is a fluorescer or a chromophore.

16. The test device of claim 1 wherein the detectable chemical group in the labeled reagent possesses a detectable chemical property and the detection zone is incorporated with a detectant composition which interacts with the chemical group to provide the detectable signal.

17. The test device of claim 16 wherein the detectable chemical group is (i) an enzyme or (ii) a substrate or cofactor for such enzyme, and wherein the detectant composition comprises the other thereof.

18. The test device of claim 17 wherein the substrate is chromogenic fluorogenic or chemiluminescent.

19. The test device of claim 1 wherein the reagent zone, reaction zone and detection zone are in the form of layers in fluid contact with one another.

20. The test device of claim 19 which additionally comprises a solid, nonporous support element situated on the opposite side of the detection layer from the reagent layer.