Hydrophilic chemilumescent acridinium labeling reagents

• US 6,924,154 B2
• Filed: 08/20/2002
• Issued: 08/02/2005
• Est. Priority Date: 08/20/2002
• Status: Expired due to Fees

• Current Assignee: Quest Diagnostic Investment Corporation, Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Original Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Inventors: Evangelista, Ramon, Garrity, Martha
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US10/225,084
• Publication Number:
  US 20040038294A1
• Time in Patent Office: 1,078 Days
• Field of Search: 546/102, 546/104, 436/500, 436/526, 436/544, 436/546, 435/6, 530/391.5
• US Class Current: 436/546
• CPC Class Codes: C07D 219/04 with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C09B 15/00 Acridine dyes
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

In accordance with the present invention, it has been discovered that introduction of hydrophilic sulfoalkyl substituents and/or hydrophilic linkers derived from homocysteic acid, cysteic acid, glycine peptides, tetraethylene oxide, and the like, offset the hydrophobicity of the acridinium ring system to produce a more soluble label which can be attached to an antibody at higher loading before precipitation and aggregation problems are encountered. Additional compounds described herein contain linkers derived from short peptides and tetraethylene oxide which increase aqueous solubility due to hydrogen bonding with water molecules. The present invention also embraces reagents for multiple acridinium labeling for signal amplification composed of a peptide bearing several acridinium esters with sulfonate groups at regularly spaced intervals for increased solubility. The invention also embraces assays employing the above-described compounds.

Claims

1. A chemiluminescent compound having the structure:

wherein;

X=O, S or NR′

, wherein R′

is H, alkyl or substituted alkyl;

Y=O or S;

Z=sulfoalkyl or sulfoalkenyl;

Ar=heteroaryl bearing at least one —

SO₂L substituent, wherein L is halogen;

R=sulfoalkyl or sulfoalkenyl;

n=0-3.

2. A compound according to claim 1 wherein X is O.

3. A compound according to claim 1 wherein Y is O.

4. A compound according to claim 1 wherein Z has the structure:

—

(CR″

₂)_q—

SO₃⁻

,

wherein;

each R″

is independently H, lower alkyl or substituted lower alkyl; and

q=1-6.

5. A compound according to claim 4 wherein Z is sulfopropyl.

6. A compound according to claim 1 wherein Z has the structure:

—

(CR″

₂)_r—

C(R″

)═

C(R″

)—

(CR″

₂)_r—

SO₃⁻

,

wherein;

each R″

is independently H, lower alkyl or substituted lower alkyl; and

each r is independently 0-4.

7. A compound according to claim 1 wherein said heteroaryl is 2,6-dialkyl substituted.

8. A compound according to claim 1 wherein said heteroaryl is a pyridyl, pyrimidyl, pyrazinyl, triazolyl, isooxazolyl, isothioazolyl, or imidazolyl.

9. A compound according to claim 8 wherein Z is sulfopropyl.

10. A chemiluminescent conjugate comprising a chemiluminescent compound according to claim 1 conjugated with a specific binding material.

11. A conjugate according to claim 10 wherein said specific binding material is an antibody.

12. A chemiluminescent assay kit comprising a conjugate according to claim 10.

13. An assay for the presence of an analyte in a sample, said assay comprising:

contacting an analyte with a conjugate according to claim 10 wherein said conjugate specifically binds to the analyte,

inducing chemiluminescence by decay of an intermediate formable from the chemiluminescent compound of the conjugate in the presence of a peroxide or molecular oxygen, and

measuring chemiluminescence therefrom to assay the analyte.

14. In a diagnostic assay for the detection of an analyte by chemiluminescence using a chemiluminescent label conjugated to a specific binding material which specifically binds to the analyte, the improvement comprising employing a compound according to claim 1 as the chemiluminescent label compound.

15. A chemiluminescent compound having the structure:

X is O,

Y is O,

Z is sulfoalkyl, and

Ar is 2,6-dimethyl-3-chlorosulfophenyl or 2,6-dimethyl-4-chlorosulfophenyl.

16. A compound according to claim 15 wherein Z has the structure:

—

(CR″

₂)_q—

SO₃,

wherein;

each R″

is independently H, lower alkyl or substituted lower alkyl; and

q=1-6.

17. A compound according to claim 15 wherein Z is sulfopropyl.

18. A compound according to claim 17 wherein the compound is 2,6-(dimethyl)-3-chlorosulfophenyl-N-(3-sulfopropyl)-acridinium-9-carboxylate.

19. A compound according to claim 17 wherein the compound is 2,6-(dimethyl)-4-chlorosulfophenyl-N-(3-sulfopropyl)-acridinium-9-carboxylate.

20. A chemiluminescent conjugate comprising a chemiluminescent compound according to claim 15 conjugated with a specific binding material.

21. A conjugate according to claim 20 wherein said specific binding material is an antibody.

22. A chemiluminescent assay kit comprising a conjugate according to claim 20.

23. An assay for the presence of an analyte in a sample, said assay comprising:

contacting an analyte with a conjugate according to claim 20 wherein said conjugate specifically binds to the analyte,

inducing chemiluminescence by decay of an intermediate formable from the chemiluminescent compound of the conjugate in the presence of a peroxide or molecular oxygen, and

measuring chemiluminescence therefrom to assay the analyte.

24. In a diagnostic assay for the detection of an analyte by chemiluminescence using a chemiluminescent label conjugated to a specific binding material which specifically binds to the analyte, the improvement comprising employing a compound according to claim 15 as the chemiluminescent label compound.

25. A compound having the structure:

wherein;

X=O, S or NR′

, wherein R′

is H, alkyl or substituted alkyl;

Y=O or S;

Z=sulfoalkyl or sulfoalkenyl;

Ar=aryl or heteroaryl bearing at least one —

SO₂L substitutent, wherein L is halogen;

R=sulfoalkyl or sulfoalkenyl; and

n=1-3.

26. A chemiluminescent conjugate comprising a chemiluminescent compound according to claim 25 conjugated with a specific binding material.

27. A conjugate according to claim 26 wherein said specific binding material in an antibody.

28. A chemiluminescent assay kit comprising a conjugate according to claim 26.

29. An assay for the presence of an analyte in a sample, said assay comprising:

contacting an analyte with a conjugate according to claim 26 wherein said conjugate specifically binds to the analyte,

inducing chemiluminescence by decay of an intermediate formable from the chemiluminescent compound of the conjugate in the presence of a peroxide or molecular oxygen, and

measuring chemiluminescence therefrom to assay the analyte.

30. In a diagnostic assay for the detection of an analyte by chemiluminescence using a chemiluminescent label conjugated to a specific binding material which specifically binds to the analyte, the improvement comprising employing a compound according to claim 25 as the chemiluminescent label compound.