Concentrating zone method in heterogeneous immunoassays

• US 4,366,241 A
• Filed: 08/07/1980
• Issued: 12/28/1982
• Est. Priority Date: 08/07/1980
• Status: Expired due to Term

• Current Assignee: Dade Behring Marburg GmbH (Siemens AG)
• Original Assignee: Syva Co. (Siemens AG)
• Inventors: Tom, Henry K., Rowley, Gerald L.
• Primary Examiner(s): Wiseman, Thomas G.
• Application Number:
  US06/176,177
• Time in Patent Office: 873 Days
• Field of Search: 435/5, 435/7, 435/188, 435/805, 435/810, 422/55, 422/56, 422/61, 23/230 B, 23/230 R, 424/1, 424/8, 424/12
• US Class Current: 435/7.91
• 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 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures

Abstract

Method and apparatus are provided for performing immunoassays employing a device comprising a relatively small test zone referred to as an immunosorbing zone, and a relatively large liquid absorbing zone in liquid receiving relationship with said immunosorbing zone. The immunosorbing zone includes a member of an immunological pair ("mip")--ligand and antiligand--bound to a support.

A signal producing system is employed in conjunction with said device having as one component a signal label bound to a mip. The signal producing system provides for production of a detectible signal in the immunosorbing zone in relation to the amount of analyte in a sample.

Claims

1. An immunoassay method for determining an analyte which is a member of an immunological pair, defined as a mip, consisting of ligand and its homologous antiligand, said method employing in combination as assay device and a signal producing system;

said assay device characterized by an immunosorbing zone comprising mip non-diffusively bound to at least a portion of a bibulous support serving as an inlet port for liquids into said device; and

a liquid absorbing zone in liquid receiving relationship with said immunosorbing zone; and

a signal producing system characterized by being capable of producing a detectible signal in said immunosorbing zone and having one component conjugated to a mip to provide a signal label-mip conjugate, wherein the amount of signal label producing said detectible signal in said immunosorbing zone is related to the amount of analyte;

said method comprising;

contacting said assay device in a predetermined order with;

(a) a solution of a sample suspected of containing said analyte; and

(b) a solution of components of said signal producing system other than components bound to said assay device, wherein said immunosorbing zone is immersed in said sample solution;

flowing said sample solution of substantially constant composition through said immunosorbing zone;

whereby said solutions migrate through said immunosorbing zone into said liquid absorbing zone resulting in an amount of signal label-mip conjugate becoming bound to said mip bound to said support in relation to the amount of analyte in said sample and affording production of a detectable signal in said immunosorbing zone; and

determining said detectable signal.

2. A method according to claim 1, wherein said signal label-mip conjugate is contacted with said assay device in a solution not earlier than concurrently with said sample solution.

3. A method according to claim 2, wherein said solution of said sample and said solution of said signal label-mip conjugate are combined prior to contacting with said assay device, with the proviso that where no additional solution of components of said signal producing system is combined with said assay device, said assay device is contacted with a wash solution subsequent to contacting with said sample and signal label solution.

4. A method according to claim 2, wherein said assay device is contacted with said solution of said sample in a first step;

said assay device is contacted with a solution of said signal label-mip conjugate in a second step; and

said assay device is contacted with remaining components of said signal producing system in a third step.

5. A method according to claim 1, wherein said signal label-mip conjugate is bound to said mip in said immunosorbing zone, wherein one of the mips is a monoepitopic ligand.

6. A method according to claim 5, where said assay device is contacted with said solution of said sample in a first step, and said assay device is contacted with said solution of components of said signal producing system in a subsequent step.

7. A method according to claims 1, 2, or 5, wherein said signal label is a fluorescer.

8. A method according to claims 1, 2 or 5, wherein said signal label is a radioisotope.

9. A method according to claim 7, wherein said analyte is a monoepitopic ligand.

10. A method according to claim 7, wherein said analyte is an antigen ligand.

11. A method according to claims 1, 2 or 5, wherein said signal label is an enzyme.

12. A method according to claim 11, wherein said analyte is a monoepitopic ligand.

13. A method according to claim 11, wherein said analyte is an antigen ligand.

14. A method according to claims 1, 2 or 5, wherein said analyte is an immunoglobulin.

15. An immunoassay method for determining an analyte which is a member of an immunological pair, defined as a mip, consisting of ligand and its homologous antiligand, said method employing in combination an assay device and a signal producing system;

said assay device characterized by an immunosorbing zone comprising mip non-diffusively bound to at least a portion of a bibulous support serving as an inlet port for liquids into said device; and

a liquid absorbing zone in liquid receiving relationship with said immunosorbing zone and at least partially enclosed to prohibit contact with liquids except through said immunosorbing zone;

said signal producing system characterized by being capable of producing a detectible signal in said immunosorbing zone and having (1) an enzyme as a signal label conjugated to a mip to provide an enzyme-mip conjugate, wherein the amount of enzyme producing said detectible signal in said immunosorbing zone is related to the amount of analyte, and (2) a signal generator precursor which is enzymatically transformed to a signal generator;

said method comprising;

contacting said assay device in a predetermined order with;

(a) a solution of a sample suspected of containing said analyte;

(b) a solution of enzyme-mip conjugate, except when said enzyme-mip conjugate is previously bound to said assay device through complexation with said mip bound to said support; and

(c) a solution of a signal generator precursor,

wherein said immunosorbing zone is substantially completely immersed in said sample solution;

flowing said sample solution having substantially constant composition through said immunosorbing zone;

whereby said solutions migrate through said immunosorbing zone into said liquid absorbing zone and said signal generator precursor is converted by said enzyme to a signal generator producing a detectible signal in said immunosorbing zone; and

determining said detectable signal.

16. A method according to claim 15, wherein a second enzyme is non-diffusively bound in said immunosorbing zone, wherein the product from one enzyme is a substrate for the other enzyme.

17. A method according to claim 16, wherein said second enzyme produces a product which is a substrate of said enzyme-mip conjugate.

18. A method according to claim 16, wherein said enzyme-mip conjugate and said signal generator precursor are combined prior to combining with said assay device.

19. A method according to claim 16 or 17, wherein said mip is bound to only a portion of said support and said second enzyme and antienzyme for said enzyme-mip conjugate are bound in a detection zone in said immunosorbing zone downstream from said mip.

20. A method according to claims 16 or 17, wherein said second enzyme is glucose oxidase and said enzyme-mip conjugate is horseradish peroxidase.

21. A method according to claim 18 wherein said assay device is contacted with said solution of said sample in a first step, and said assay device is contacted with said combined enzyme-mip conjugate and signal generator precursor in a second step.

22. An immunoassay method for determining an analyte which is a member of an immunological pair, defined as a mip, consisting of ligand and its homologous antiligand, said method employing in combination an assay device and a signal producing system;

said assay device characterized by an immunosorbing zone comprising mip non-diffusively bound to a bibulous support serving as a inlet port for liquids into said device; and

a liquid absorbing zone in liquid receiving relationship with said immunosorbing zone and at least partially enclosed to prohibit contact with liquids except through said immunosorbing zone;

said signal producing system characterized by being capable of producing a detectible signal in said immunosorbing zone and having a fluorescer as signal label conjugated to a mip to provide a fluorescer-mip conjugate, wherein the amount of fluorescer producing said detectible signal in said immunosorbing zone is related to the amount of analyte;

said method comprising;

contacting said assay device in a predetermined order with;

(a) a solution of a sample suspected of containing said analyte;

(b) a solution of fluorescer-mip conjugate, except when said fluorescer-mip conjugate is bound to said assay device prior to combining with said sample solution; and

(c) a wash solution, except when said fluorescer-mip conjugate is bound to said assay device prior to combining with said sample solution,

whereby said solutions migrate through said immunosorbing zone into said liquid absorbing zone and said fluorescer-mip conjugate is bound in said immunosorbing zone in relation to the amount of analyte in said sample; and

determining the fluorescence from said immunosorbing zone.

23. A method according to claim 22, wherein said fluorescer-mip conjugate is bound to said assay device before contacting with said sample solution.

24. A method according to claim 22, wherein said fluorescer-mip conjugate is combined with said assay device after said sample solution is combined with said assay device.

25. An assay device for performing immunoassays which comprises:

an immunosorbing member comprising a member of an immunological pair, defined as a mip, non-diffusively bound to at least a portion of a bibulous support; and

a liquid absorbing member comprising a bibulous material in liquid receiving relationship with said immunosorbing member and extending transversely therefrom, wherein at least the portion of said liquid absorbing member about said immunosorbing member is enclosed in an impermeable enclosure to inhibit contact with solutions except through said immunosorbing member and said immunosorbing member has a relatively small liquid holding capacity as compared to said liquid absorbing member.

26. An assay device according to claim 25, wherein said immunosorbing member and said liquid absorbing member are a single strip, said immunosorbing member being at the end of said single strip.

27. An assay device according to claim 25, wherein said immunosorbing member is a layer above a portion of said liquid absorbing member.

28. An assay device according to claims 25, 26, or 27, wherein bound to said bilbulous support is an enzyme.

29. An assay device according to claims 25, 26, or 27, wherein bound to said mip is a labeled form of the homologous mip, substantially saturating all of the binding sites of said mip bound to said support.

32. An immunoassay kit comprising an assay device according to claims 25, 26, 27, or 30 in combination with a labeled mip separate from said assay device, wherein said labeled mip is the same as or complementary to said mip bound to said bibulous support, said labeled mip and said bound mip are present in amounts to substantially optimize the signal observed at said immunosorbing member.

33. A kit according to claim 32, wherein said label is an enzyme and including in combination, an enzyme substrate capable of providing a fluorescent or visible light absorptive product.

34. A kit according to claim 33, wherein said labeled mip is radioactively labeled mip.

30. An assay device for performing immunoassay comprising:

(1) an immunosorbing member;

(2) a liquid absorbing member;

(3) a non-permeable barrier;

said immunosorbing member serving as a liquid entry port to said device and comprised of at least two zones;

a first bibulous layer to which a member of an immunological pair, defined as mip, is non-diffusively bound, and a second zone between said bibulous member and said liquid absorbing member of different flow resistant characteristic from said bibulous layer;

said liquid absorbing member comprised of a bibulous layer in liquid receiving relationship with said immunosorbing zone and extending transversely therefrom, wherein said immunosorbing member has relatively small liquid holding capacity as compared to said liquid absorbing member; and

said non-permeable barrier comprising a non-permeable layer about said liquid absorbing and immunosorbing members to inhibit entry of liquid to said device except through said immunosorbing member.

31. An assay device according to claim 30, wherein said second zone is substantially non-resistive to liquid flow.