Analyte-substitute reagent for use in specific binding assay methods, devices and kits

• US 5,340,748 A
• Filed: 05/25/1993
• Issued: 08/23/1994
• Est. Priority Date: 07/18/1990
• Status: Expired due to Fees

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Baugher, Bennett W., Devereaux, Sharon M., Chamberlain, Aurora J., Ungemach, Frank S.
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Wolski, Susan C.
• Application Number:
  US08/067,254
• Time in Patent Office: 455 Days
• Field of Search: 436/514, 436/518, 436/537, 436/538, 436/540, 436/816, 436/822, 436/817, 436/539, 436/541, 435/7.92, 435/7.93, 435/971, 435/973, 435/975
• US Class Current: 436/518
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/946 CNS-stimulants, e.g. cocaine, amphetamines
• CPC Class Codes: G01N 33/948 Sedatives, e.g. cannabinoids, barbiturates opiates G01N33/9486
• CPC Class Codes: G01N 33/9486 Analgesics, e.g. opiates, aspirine
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/816 Alkaloids, amphetamines, and barbiturates
• CPC Class Codes: Y10S 436/817 Steroids or hormones
• CPC Class Codes: Y10S 436/822 Identified hapten

Abstract

Assay reagents, devices, methods and kits used in the analysis of low molecular weight analytes which by themselves are too small or unable to bind to two specific binding members at the same time. The invention involves the use of an analyte-substitute reagent (ASR) comprising at least two components, the first of which is identical to or an analog of the analyte to be determined, while the second is an unrelated ligand for which an antibody or other specific binding member can be obtained or produced.

Claims

1. A method for determining the presence or amount of an analyte of interest in a test sample, comprising the steps of:

contacting said test sample sequentially or simultaneously with

(i) an analyte-substitute reagent comprising an analyte-component conjugated to a ligand-component,

wherein said analyte-component has at least one epitope in common with said analyte of interest, and said analyte-component specifically binds to a single analyte-specific binding member, and

wherein said ligand-component specifically binds to a ligand-specific binding member but does not specifically bind to an analyte-specific binding member, and

(ii) a first analyte-specific binding member which specifically binds an epitope present on both said analyte of interest and said analyte-component of said analyte-substitute reagent,

thereby forming in the presence of said analyte of interest, a mixture of;

analyte of interest/first analyte-specific binding member complex;

analyte-substitute reagent/first analyte-specific binding member complex; and

unbound analyte-substitute reagent;

wherein the amount of unbound analyte-substitute reagent increases as the amount of said analyte of interest increases;

contacting said mixture sequentially or simultaneously with

(i) a capture reagent comprising a second specific binding member which specifically binds said analyte-substitute reagent, wherein said capture reagent is directly or indirectly bound to a solid phase, and

(ii) an indicator reagent comprising a label capable of producing a detectable signal, wherein said label is conjugated to a third specific binding member which directly or indirectly specifically binds said unbound analyte-substitute reagent,

wherein said capture reagent and said indicator reagent form a directly or indirectly bound detectable complex with said unbound analyte-substitute reagent and wherein when said capture reagent specifically binds said analyte-component then said indicator reagent specifically binds said ligand-component, and when said capture reagent specifically binds said ligand-component then said indicator reagent specifically binds said analyte-component; and

(c) separating said directly or indirectly bound detectable complex from the mixture and detecting said label associated with said detectable complex, or the amount of said indicator reagent that is not associated with said detectable complex, to determine the presence or amount of said analyte of interest.

2. The method according to claim 1, wherein said capture reagent specifically binds said ligand-component of said analyte-substitute reagent, and said indicator reagent specifically binds said analyte-component of said analyte-substitute reagent.

3. The method according to claim 1, wherein said analyte-substitute reagent is prebound to said first specific binding member as an analyte-substitute reagent/first specific binding member complex, and wherein said analyte-substitute reagent is displaced from said analyte-substitute reagent/first specific binding member complex when analyte is present in the test sample.

4. The method according to claim 1, wherein said capture reagent specifically binds said analyte-component of said analyte-substitute reagent, and said indicator reagent specifically binds said ligand-component of said analyte-substitute reagent.

5. The method according to claim 1, further comprising, contacting said label with at least one additional signal generating substance capable of reacting with said label to produce said detectable signal.

6. The method according to claim 1, wherein said first specific binding member and said analyte-substitute reagent are present as a tablet, capsule, powder or liquid reagent.

7. The method according to claim 1, wherein said capture reagent is directly immobilized upon a solid phase material.

8. The method according to claim 1, further comprising the step of directly immobilizing said capture reagent upon a solid phase material.

9. The method according to claim 1, further comprising the addition of at least one ancillary specific binding member to indirectly bind said indicator reagent to said analyte-substitute reagent.

10. The method according to claim 1, further comprising the addition of at least one ancillary specific binding member to indirectly bind said capture reagent to a solid phase.

21. The method according to claim 1, wherein said capture reagent is indirectly bound to a solid phase through an ancillary specific binding member which specifically binds to said capture reagent and is itself immobilized upon a solid phase, thereby immobilizing said capture reagent and complexes thereof.

11. A kit for determining the presence or amount of an analyte of interest in a test sample, comprising a packaged combination of:

(a) at least one analyte-substitute reagent, each of which comprises an analyte-component conjugated to a ligand-component,

wherein said analyte-component has at least one epitope in common with one analyte or interest, and said analyte-component specifically binds to a single analyte-specific binding member, and

wherein said ligand-component specifically binds to a ligand-specific binding member but does not specifically bind an analyte-specific binding member;

(b) at least one first analyte-specific binding member, each of which specifically binds an epitope present on both a single analyte of interest and a single analyte-substitute reagent;

(c) at least one capture reagent, each of which comprises a second specific binding member which specifically binds to either said analyte-component or said ligand-component of said at least one analyte-substitute reagent wherein either said capture reagent is directly bound to a solid phase, or said kit further comprises at least one ancillary binding member each of which specifically bind said at least one capture reagent and each of which are immobilized upon a solid phase; and

(d) at least one indicator reagent, each of which comprises a label capable of producing a detectable signal, wherein said label is conjugated to a third specific binding member which directly or indirectly specifically binds said at least one analyte-substitute reagent,

wherein when said capture reagent specifically binds said analyte-component then said indicator reagent specifically binds said ligand-component, and when said capture reagent specifically binds said ligand-component then said indicator reagent specifically binds said analyte-component.

12. The kit according to claim 11, further comprising at least one additional signal generating substance which is capable of reacting with said label to produce a detectable signal.

13. The kit according to claim 11, wherein said at least one capture reagent comprises a second specific binding member each of which specifically binds said analyte-component of said at least one analyte-substitute reagent and said at least one indicator reagent comprises a third specific binding member each of which specifically binds said ligand-component of said at least one analyte-substitute reagent.

14. The kit according to claim 11, wherein said at least one capture reagent comprises a second binding member each of which specifically binds said ligand-component of said at least one analyte-substitute reagent, and said at least one indicator reagent comprises a third binding member each of which specifically binds said analyte-component of said at least one analyte-substitute reagent.

15. The kit according to claim 11, wherein said at least one first analyte-specific binding member and said at least one analyte-substitute reagent are present as a tablet, capsule, powder or liquid reagent.

16. The kit according to claim 11, wherein said at least one capture reagent is immobilized on a solid phase material.

17. The kit according to claim 16, wherein said at least one first specific binding member, said at least one analyte-substitute reagent, said at least one capture reagent and said at least one indicator reagent are present on said solid phase, said solid phase comprising a sufficient number of zones or layers to contain said reagents, wherein a first zone contains said at least one first specific binding member such that analyte-substitute reagent/first specific binding member complex is formed prior to the contact of the test sample with a second zone containing said at least one capture reagent.

18. The kit according to claim 11, further comprising at least one ancillary binding member each of which specifically bind said at least one capture reagent and each of which are immobilized upon a solid phase.

19. The kit according to claim 11, comprising a plurality of analyte-substitute reagents and a single capture reagent, wherein each analyte-substitute reagent has a different analyte-component and a common ligand-component and said single capture reagent specifically binds to said common ligand-component of said analyte-substitute reagents.

20. The kit according to claim 11, comprising a plurality of analyte-substitute reagents and a plurality of capture reagents, wherein each analyte-substitute reagent has a different analyte-component and a different ligand-component, and each capture reagent specifically binds to one of said different ligand-components of said analyte-substitute reagents.

22. A method for determining the presence or amount of a plurality of analytes of interest in a test sample, comprising the steps of:

(a) contacting said test sample sequentially or simultaneously with

(i) a plurality of analyte-substitute reagents, each analyte-substitute reagent comprising an analyte-component conjugated to a ligand-component,

wherein said analyte-component has at least one epitope in common with one analyte of interest in the test sample, and said analyte-component specifically binds a single analyte-specific binding member, and

wherein said ligand-component specifically binds a single ligand-specific binding member but does not specifically bind an analyte-specific binding member, and

(ii) a plurality of first analyte-specific binding members, each of which specifically binds an epitope present on both said one analyte of interest and said analyte-component of said analyte-substitute reagent,

thereby forming in the presence of said analytes of interest, a mixture of;

analyte of interest/first analyte-specific binding member complexes;

analyte-substitute reagent/first analyte-specific binding member complexes; and

unbound analyte-substitute reagents, wherein the amount of unbound analyte-substitute reagents increases as the amount of analytes of interest in the test sample increases;

contacting said mixture sequentially or simultaneously with

(i) at least one capture reagent comprising a second specific binding member which specifically binds said analyte-substitute reagents, wherein said capture reagent is directly or indirectly bound to a solid phase, and

(ii) at least one indicator reagent comprising a label capable of producing a detectable signal, wherein said label is conjugated to a third specific binding member which directly or indirectly specifically binds said unbound analyte-substitute reagents,

wherein said at least one capture reagent and said at least one indicator reagent for directly or indirectly bound detectable complexes with said unbound analyte-substitute reagents and wherein when said capture reagents specifically bind said analyte-components then said indicator reagents specifically bind said ligand-components, and when said capture reagents specifically bind said ligand-components then said indicator reagents specifically bind said analyte-components; and

(c) separating said directly or indirectly bound detectable complexes from the mixture and detecting said label associated with said detectable complexes, or the amount of said at least one indicator reagent that is not associated with said detectable complexes, to determine the presence or amount of said analytes of interest in the test sample.

23. The method according to claim 22, wherein a plurality of analytes of interest are assayed using a plurality of analyte-substitute reagents and a plurality of capture reagents, wherein each analyte-substitute reagent has a different analyte-component and a different ligand-component, and each capture reagent specifically binds to one of said different ligand-components of said analyte-substitute reagents.

24. The method according to claim 22, wherein a plurality of analytes of interest are assayed using a plurality of analyte-substitute reagents and a single capture reagent, wherein each analyte-substitute reagent has a different analyte-component and a common ligand-component, and said single capture reagent specifically binds to said common ligand-component of said analyte-substitute reagents.