Method for determining an analyte in a liquid sample using a zoned test device and an inhibitor for a label used in said method

• US 5,104,793 A
• Filed: 02/16/1988
• Issued: 04/14/1992
• Est. Priority Date: 02/16/1988
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics Corporation (Roche Holding AG)
• Original Assignee: Boehringer Mannheim Corporation
• Inventors: Buck, Harvey
• Primary Examiner(s): SAUNDERS, DAVID A
• Application Number:
  US07/156,168
• Time in Patent Office: 1,519 Days
• Field of Search: 435/7, 435/805, 435/810, 435/7.92, 435/7.94, 435/963, 435/970, 435/971, 436/512, 436/514, 436/518, 436/548, 436/530, 436/800, 436/808, 436/810, 422/56-58, 422/55, 422/69
• US Class Current: 435/7.92
• 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: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/963 Methods of stopping an enzyme reaction or stabilizing the test materials
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

The invention relates to a heterogeneous immunoassay which is carried out using a multizoned test device. In particular, the invention involves the use of an inhibitor of a label which is used in the assay. The label which may be an enzyme, is attached to a receptor such as an antibody. The inhibitor is not acted upon by the label, but must be removed in order for a signal to be produced. Also described are test strips which can be used for the assay.

Claims

1. Method for determining an analyte in a liquid sample, comprising:

(i) contacting a liquid sample to a zoned device containing first, second, and third zones, wherein said first and second zones are in fluid contact with each other and said second and third zones are in fluid contact with each other, to permit flow of liquid sample from said first zone into said second zone and from said second zone into said third zone, wherein

(ii) said first zone contains at least one non-solid phase bound, label carrying receptor which specifically binds to said analyte to form a complex of receptor and analyte in a liquid phase,

(iii) said second zone contains a non-solid phase bound inhibitor for the label of said labelled receptor, wherein said inhibitor is not acted upon by said label and directly inhibits said label from forming a detectable signal when said liquid phase containing complexes of receptor and analyte flows from said first zone and into said second zone,

(iv) said third zone contains a solid phase bound, unlabelled receptor which specifically binds to complexes of analyte and labelled receptor but not to said inhibitor when liquid containing (a) complexes of analyte and labelled receptor and (b) inhibitor flow therein from said second zone, wherein said contacting is carried out under conditions favoring binding of said complex and said solid phase receptor,

(v) removing said inhibitor from said third zone, and

(vi) determining a detectable signal produced in said third zone by said label in said complex as a determination of said analyte.

2. Method of claim 1, wherein said receptor is an antibody or fragment thereof.

3. Method of claim 1, wherein said receptor is a monoclonal antibody or fragment thereof.

4. Method of claim 1, wherein said label is an enzyme.

5. Method of claim 4, wherein said inhibitor is an analogue of a substrate for said enzyme.

6. Method of claim 1, wherein said label is a fluorescent label.

7. Method of claim 6, wherein said inhibitor is a quenching molecule for said label.

8. Method of claim 1, wherein treating said complex to remove said inhibitor comprises washing said complex with a fluid.

9. Method of claim 1 further comprising contacting said complex with a substance which competes with said inhibitor for a binding site on said label in an amount sufficient to displace at least a portion of said inhibitor.

10. Method of claim 1, wherein said first zone further comprises a second, non-solid phase bound unlabelled receptor which specifically binds to said analyte but not said labelled receptor, and said solid phase bound, unlabelled receptor specifically binds to said second, non solid phase bound, unlabelled receptor.

12. Method of claim 1, wherein said analyte is contacted with a plurality of receptors one of which carries a label with an inhibitor specific for that label carried thereon.

13. Method of claim 12, wherein one of said plurality of receptors comprises a labeled Fab fragment.

14. Method of claim 12, wherein said plurality of receptors comprises monoclonal antibodies or fragments thereof.

11. Method for determining an analyte in a liquid sample comprising:

(i) contacting a liquid sample to a zoned device containing a first and second zone which are in fluid contact with each other so as to permit flow of liquid from said first zone to said second zone, wherein

(ii) said first zone contains both (a) at least one non-solid phase bound, labelled receptor which specifically binds to said analyte to form a complex of receptor and analyte upon contact with analyte containing liquid and (b) a non-solid phase bound inhibitor for the label of said labelled receptor, wherein said inhibitor is not acted upon by said label and directly inhibits said label from providing a detectable signal,

(iii) said second zone contains a solid phase bound, unlabelled receptor which specifically binds to complexes of analyte and labelled receptor but not to said inhibitor when liquid containing (a) complexes of analyte and labelled receptor and (b) inhibitor flow therein from said first zone,

wherein said contacting is carried out under conditions favoring binding of said complex and said solid phase receptor,

(iv) removing said inhibitor from said second zone, and

(v) determining a detectable signal produced in said second zone by said label in said complex as a determination of said analyte.

15. Method of claim 11, wherein said first zone further comprises a second, non-solid phase bound unlabelled receptor which specifically binds to said analyte but not said unlabelled receptor, and said solid phase bound, unlabelled receptor specifically binds to said second, non solid phase bound, unlabelled receptor.

16. Method of claim 11, wherein said label is an enzyme.

17. Multizone test apparatus useful in determining an analyte in a liquid sample, said test apparatus comprising a first zone in fluid contact with a second zone, and a third zone in fluid contact with said second zone, wherein:

(i) said first zone contains at least one non-solid phase bound, label carrying receptor which specifically binds to said analyte,

(ii) said second zone contains a non-solid phase bound inhibitor for the label of said unlabelled receptor, wherein said inhibitor is not acted upon by said label and directly inhibits said label from providing a detectable signal, and

(iii) said third zone contains a solid phase bound, unlabelled receptor which specifically binds to a complex of said analyte and said first receptor.

18. Multizone test apparatus of claim 17, wherein said first zone further comprises a second, non-solid phase bound, unlabelled receptor which specifically binds to said analyte but not said labelled receptor, and said solid phase bound, unlabelled receptor binds to a complex of analyte, labelled receptor and unlabelled, non-solid phase bound receptor via said non-solid phase bound unlabelled receptor.

19. Multizone test apparatus of claim 17, further comprising a fourth zone positioned between said first zone and said second zone which contains a substrate which reacts with said label to form a detachable signal.

20. Multizone test apparatus of claim 17, wherein said label is an enzyme.

21. Multizone test apparatus useful in determining an analyte in a liquid sample, said test apparatus comprising a first zone in fluid contact with a second zone, wherein:

(i) said first zone contains both at least one non-solid phase bound, labelled receptor which specifically binds to said analyte, and (b) a non-solid phase bound inhibitor for the label of said labelled receptor, wherein said inhibitor is not acted upon by said label and directly inhibits said label from providing a detectable signal, and

(ii) a second zone containing a solid phase bound, unlabelled receptor which specifically binds to a complex of said analyte and said labelled receptor.

22. Multizone test apparatus of claim 21, wherein said first zone also contains a non-solid phase bound, unlabelled receptor which specifically binds to said analyte but not said non-solid phase bound, labelled receptor, and said solid phase bound, unlabelled receptor binds to a complex of analyte, labelled receptors and non-solid phase bound, unlabelled receptor via said non-solid phase bound unlabelled receptor.

23. Multizone test apparatus of claim 21, further comprising a third zone positioned between said first zone and said second zone which contains a substrate which reacts with said label to form a detectable signal.

24. Multizone test apparatus of claim 21, wherein said label is an enzyme.