Chromogenic tracers for use in an assay

• US 4,489,165 A
• Filed: 01/24/1983
• Issued: 12/18/1984
• Est. Priority Date: 01/24/1983
• Status: Expired due to Fees

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Wagner, Daniel B., Tyson, Richard L.
• Primary Examiner(s): Nucker, Christine M.
• Application Number:
  US06/460,141
• Time in Patent Office: 694 Days
• Field of Search: 436/500, 436/537, 436/546, 436/800, 436/811, 436/816, 436/817
• US Class Current: 436/500
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/78 Thyroid gland hormones , e.g. T3, T4, TBH, TBG or their receptors
• CPC Class Codes: G01N 33/9453 Cardioregulators, e.g. antihypotensives, antiarrhythmics
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function
• CPC Class Codes: Y10S 436/816 Alkaloids, amphetamines, and barbiturates
• CPC Class Codes: Y10S 436/817 Steroids or hormones

Abstract

A chromogen, in particular, a fluorescent compound, is conjugated to a ligand through an amino acid spacer radical, which includes a free carboxyl group. The tracers are particularly suitable for use in a flow through assay for low molecular weight analytes. The tracers are rapidly bound, of good sensitivity, and low non-specific binding.

Claims

1. A tracer for use in an assay comprising:

a chromogen conjugated to a ligand through a spacer radical, said spacer radical for conjugating the chromogen to the ligand being derived from an amino acid and having the following structural formula;

##STR6## wherein X is selected from the group consisting of hydrogen, ammonium, amine salt and metal cations;

Y is selected from the group consisting of --NH-- and ##STR7## and R is selected from the group consisting of substituted and unsubstituted divalent hydrocarbon radicals having no more than 13 carbon atoms and wherein --R-- and --CH-- may be linked to form a carbocyclic radical.

2. The tracer of claim 1 wherein the chromogen is a fluorescein dye.

3. The tracer of claim 2 wherein R is an alkylene group having 4 carbon atoms and Y is --NH--.

4. The tracer of claim 3 wherein the ligand is triiodothyronine.

5. The tracer of claim 3 wherein the ligand is thyroxine.

6. The tracer of claim 3 wherein the ligand is digoxigenin.

7. The tracer of claim 1 wherein R is alkylene.

8. The tracer of claim 7 wherein the chromogen is a fluorescent dye.

9. The tracer of claim 8 wherein Y is --NH--.

10. The tracer of claim 9 wherein R is an alkylene group having from 2 to 6 carbon atoms.

11. The tracer of claim 10 wherein the fluorescent dye is a fluorescein dye.

12. The tracer of claim 1 wherein the tracer is N-α

-(3-digoxigeninyl)-N-ε

-[5(6)-carboxyfluoresceinoyl]-lysine.

13. The tracer of claim 1 wherein the tracer is carboxyfluorescein-lysylamide-3-ketodigoxigenin.

14. The tracer of claim 1 wherein the tracer is carboxyfluorescein-lysylamide-3,3'"'"',5-triiodo-L-thyronine.

15. The tracer of claim 1 wherein the tracer is carboxyfluorescein-lysylamide-3,3'"'"',5-triiodo-N-acetyl-L-thyronine.

16. The tracer of claim 1 wherein the tracer is N-α

-(3-digoxigeminyl-N-ε

-(dichlorotriazinylaminofluoresceinyl)-lysine.

17. The assay of claim 1 wherein during the assay the tracer is passed through a flow through chamber including a binder for the tracer.

18. The assay of claim 17 wherein the chromogen is a fluorescein dye.

19. The assay of claim 18 wherein R is an alkylene group having 4 carbon atoms and Y is --NH--.

20. The assay of claim 19 wherein the ligand is triiodothyronine and wherein the assay is a T₃ uptake assay for T₃ serum binding.

21. The assay of claim 19 wherein the ligand is thyroxine and wherein the analyte is thyroxine.

22. The assay of claim 19 wherein the ligand is digoxigenin and wherein the analyte is digoxin.

23. The assay of claim 17 wherein R is alkylene.

24. The assay of claim 23 wherein the chromogen is a fluorescent dye.

25. The assay of claim 24 wherein Y is --NH--.

26. The assay of claim 25 wherein R is an alkylene group having from 2 to 6 carbon atoms.

27. The assay of claim 26 wherein the fluorescent dye is a fluorescein dye.

28. The assay of claim 17 wherein the tracer is carboxyfluorescein-lysylamide-N-acetyl-L-thyroxine and wherein the analyte is thyroxine.

29. The assay of claim 17 wherein the tracer is N-60 -(3-digoxigeninyl)-N-ε

-[5(6)-carboxylfluoresceinoyl]-lysine and wherein the analyte is digoxin.

30. The assay of claim 17 wherein the tracer is carboxyfluorescein-lysylamide-3,3'"'"',5-triiodo-L-thyronine and wherein the assay is a T₃ uptake assay for T₃ serum binding proteins.

31. The assay of claim 17 wherein the tracer is carboxyfluorescein-lysylamide-3,3'"'"',5-triiodo-N-acetyl-L-thyronine and wherein the assay is a T₃ uptake assay for T₃ serum binding proteins.

32. A process for determining an analyte, comprising:

contacting analyte and tracer with a binder for at least the analyte, said tracer being comprised of a chromogen conjugated to a ligand through a spacer radical, said ligand being bound by one of the analyte and the binder, said spacer radical being derived from an amino acid and having the structural formula as defined in claim 1, said contacting binding a portion of the tracer; and

determing at least one of bound and unbound tracer to determine analyte.

33. The process of claim 32 wherein the binder is a binder for both the analyte and tracer.

34. The process of claim 33 wherein bound and unbound tracer pass through a flow through chamber containing an adsorbent for the unbound tracer.

35. The process of claim 32 wherein the chromogen is a fluorescent dye.

36. The process of claim 35 wherein the analyte has a molecular weight of no greater than 2,000.

37. A reagent kit for use in an assay for an analyte, comprising:

a package, said package containing a binder for the analyte and a tracer for determining the analyte, said tracer being a tracer as defined in claim 1.

38. A regent kit for use in an essay for an analyte, comprising:

a package, said package containing a binder for the analyte and a tracer for determining the analyte, said tracer being a tracer as defined in claim 3.

39. A reagent kit for use in an assay for an analyte, comprising:

a package, said package containing a binder for the analyte and a tracer for determining the analyte, said tracer being a tracer as defined in claim 4.

40. A reagent kit for use in an assay for an analyte, comprising:

a package, said package containing a binder for the analyte and a tracer for determining the analyte, said tracer being a tracer as defined in claim 5.

41. A reagent kit for use in an assay for an analyte, comprising:

a package, said package containing a binder for the analyte and a tracer for determining the analyte, said tracer being a tracer as defined in claim 6.

42. A reagent kit for use in an assay for an analyte, comprising:

a package, said package containing a binder for the analyte and a tracer for determining the analyte, said tracer being a tracer as defined in claim 11.