Migratory detection immunoassay

• US 4,956,275 A
• Filed: 04/14/1987
• Issued: 09/11/1990
• Est. Priority Date: 04/14/1987
• Status: Expired due to Fees

• Current Assignee: Molecular Devices
• Original Assignee: Molecular Devices
• Inventors: Zuk, Robert F., Briggs, Jonathan, Armenta, Richard D.
• Primary Examiner(s): Kepplinger, Esther M.
• Assistant Examiner(s): Scheiner, Toni R.
• Application Number:
  US07/038,294
• Time in Patent Office: 1,246 Days
• Field of Search: 436/514, 436/515, 436/518, 436/807, 436/810, 435/7, 435/288, 435/291, 435/805, 435/25
• US Class Current: 435/7.92
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: G01N 33/54388 based on lateral flow
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• 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

Abstract

Novel methods and devices are provided for detecting analytes, where the sample is placed upstream from a reagent which is a component of a signal-producing system. The detection zone is provided upstream from the sample, where the amount of reagent which becomes bound at two sites is determined and the two values used for an accurate determination of an analyte.

Claims

1. A method for determining the presence of an analyte in a sample suspected of containing said analyte, said method employing a strip device capable of providing capillary transport and comprising at least four regions:

in the direction of eluent migration (1) a reagent region displaced from a first end of said strip device, containing a reagent consisting of a labeled specific binding member diffusibly bound to said strip, (2) a first detection region comprising a bound complementary specific binding member of said labeled specific binding member, said complementary specific binding member being capable of specifically binding (3) a sample region for receiving said sample; and

(4) a second detection region comprising the bound complementary specific binding member of said labeled specific binding member wherein said second detection region nay extend from said reagent region into any reservoir region;

with the proviso that said sample region and said second detection region may overlap, said method employing a signal producing system capable of producing a detectable signal at said second detection region and comprising said labeled specific binding member;

said method comprising

adding said sample to said sample region;

immersing said first end in an eluent to provide a moving liquid phase through said strip, whereby said eluent migrates through said strip carrying said labeled specific binding member and, when sample is outside said detection region, carrying sample into said detection region, and distributing said labeled specific binding member within at least one measurement site in said second detection region in relation to the amount of analyte in said sample, wherein there are at least two spatially separated measurement sites; and

determining the amount of bound labeled specific binding member by means of said signal producing system at said measurement sites as compared to a sample having a known amount of analyte, where the relationship between the amounts at the different sites is compared to the relationship obtained with a sample having a known amount of analyte.

2. A method according to claim 1, wherein said labeled specific binding member comprises an enzyme label.

3. A method according to claim 2, wherein said enzyme is an oxidoreductase.

4. A method according to claim 2, wherein a single determination is made at the sample region.

5. A method according to claim 1, wherein said complementary specific binding member is an antibody.

6. A method according to claim 5, wherein said antibody is uniformly distributed in said detection region.

7. A method according to claim 5, wherein one of said measurement sites is said sample region, where said sample region overlaps said detection region.

8. A method according to claim 1, wherein said labeled specific binding member is present in said reagent region in sufficient amount to substantially saturate the complementary specific binding member in said detection region in the presence of analyte in the sample at the maximum amount of the range of interest of said analyte.

9. A method according to claim 1, wherein said labeled specific binding member comprises an enzyme label and said eluent comprises at least one member of said signal producing system.

10. A method according to claim 9, wherein said one member of said signal producing system is a substrate or cofactor--for said enzyme.

11. A method according to claim 1, including the additional step of contacting said measurement sites with a developer solution containing additional members of said signal producing system.

12. A method according to claim 1, wherein said strip device is comprised of a plurality of bibulous materials.

13. A method according to claim 1, wherein said specific binding member is distributed uniformly through said detection region.

14. A method according to claim 1, wherein said specific binding member is distributed non-uniformly through said detection region.

29. A method according to claim 1 or 5, further comprising a reservoir region with a second end, wherein said second detection region and said reservoir region may partially overlap.

15. A method for determining the presence of an analyte in a sample suspected of containing said analyte, said method employing a strip device capable of providing capillary transport and comprising a bibulous layer with a least four regions:

in the direction of eluent migration (1) a reagent region displaced from a first end of said strip device, containing a reagent consisting of an enzyme labeled specific binding member diffusibly bound to said layer, (2) a first detection region comprising the bound complementary specific binding member of said enzyme labeled specific binding member, said complementary specific binding member being capable of specifically binding said analyte, (3) a sample region for receiving said sample; and

(4) a second detection region comprising the bound complementary specific binding member of said enzyme labeled specific binding member;

with the proviso that said sample region and said second detection region may overlap, and an inert support layer overlapping said bibulous layer and providing for access to said first end and at least two detection sites in the second detection region;

said method employing a signal producing system capable of producing a detectable signal at said detection sites and comprising said enzyme labeled specific binding member and enzyme substrate;

said method comprising;

adding said sample to said sample region;

immersing said first end in an eluent to provide a moving liquid phase through said strip, whereby said eluent migrates through said strip carrying said enzyme labeled specific binding member and, when sample is outside said second detection region, carrying sample into said second detection region, and distributing said enzyme labeled specific binding member through said second detection region in relation to the amount of analyte in said sample; and

determining the amount of bound labeled specific binding member by means of said signal producing system at least two spatially separated detection sites, where the relationship between the amounts at the different sites is compared to the relationship obtained with a sample having known amount of analyte.

16. A method according to claim 15, including the additional step of contacting said detection sites with a developer solution comprising additional members of said signal producing system to produce a signal resulting in light absorption.

17. A method according to claim 15, wherein one of said detection sites is the sample region.

18. A method according to claim 15, wherein one of said detection sites is between said reagent region and said sample region.

19. A method according to claim 15, including the additional step of contacting said detection sites with a developer solution comprising additional members of said signal producing system to produce a signal resulting in a change in pH.

20. A method according to claim 15, including the additional step of contacting said detection sites with a developer solution comprising additional members of said signal producing system to produce a signal resulting in a change in redox potential.

21. A method according to claim 15, wherein said eluent comprises additional members of the signal producing system.

22. A strip device capable of providing capillary transport and comprising a bibulous layer comprising at least four regions:

in the direction of eluent migration (1) a reagent region displaced from a first end of said strip device, containing a reagent consisting of a labeled specific binding member diffusibly bound to said strip wherein said labeled specific binding member is member of a signal producing system;

(2) a first detection region comprising the bound complementary specific binding member of said labeled specific binding member, said complementary specific binding member being capable of specifically binding an analyte;

(3) a sample region for receiving said sample; and

(4) a second detection region comprising the bound complementary specific binding member of said labeled specific binding member;

with the proviso that said sample region may overlap with any detection region, but each detection region must be spatially separated from every other detection region, and an inert support layer overlapping said bibulous layer and providing for access to said first end and said detection sites.

23. A strip device according to claim 22, wherein at least two of said regions are of different materials.

24. A strip device according to claim 22, wherein said bound complementary specific binding member is substantially uniformly distributed through said sample region and said first and second detection regions.

25. A strip device according to claim 22, wherein said labeled specific binding member comprises an enzyme label.

26. A strip device according to claim 22, wherein said sample region overlaps said first and second detection regions, and said sample region is one of said detection sites.

27. A strip device according to claim 22, wherein said first and second detection regions comprise members of said signal producing system.

28. A kit comprising a strip device according to claim 25, substrate for said enzyme and any required cofactors for said enzyme.

30. A strip device according to claim 22, further comprising a reservoir region, wherein said second detection region and said reservoir region may partially overlap.