Acridinium ester labels having hydrophilic modifiers

• US 6,664,043 B2
• Filed: 07/03/2001
• Issued: 12/16/2003
• Est. Priority Date: 07/03/2001
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Bayer Corporation (Bayer AG)
• Inventors: Jiang, Qingping, Sharpe, David, Natrajan, Anand
• Primary Examiner(s): DENTZ, BERNARD I
• Application Number:
  US09/898,381
• Publication Number:
  US 20030045716A1
• Time in Patent Office: 896 Days
• Field of Search: 546/104, 536/18.7, 435/5, 435/6, 435/7.1, 435/7.5, 530/391.5, 544/257, 544/268
• US Class Current: 435/5
• CPC Class Codes: G01N 33/533 with fluorescent label

Abstract

The present invention is generally directed to detectable chemiluminescent acridinium ester labels having hydrophilic modifiers; to compositions, complexes and/or conjugates which include such labels; and to processes for performing bioanalytical assays for target analytes which use such labels. Assays for folate, theophylline, and tobramycin (using such labels with hydrophilic modifiers such as nonionic polyethylene glycol and polyionic spermine disulfonate and polyionic spermine dicarboxylate) are described in detail.

Claims

1. A conjugate with improved hydrophilicity having the general structure A—

B—

C, wherein,

A is an analyte;

B is a hydrophilic modifier selected from the group consisting of polyethylene glycol having a molecular weight of about 280-5000, or a polyionic spacer derived from a polyamine where the internal, but not necessarily all the amines have been modified by hydrophilic molecules; and

C is a chemiluminescent acridinium compound containing a phenyl moiety with an ester linkage wherein the hydrophilic modifier is conjugated meta or para to the ester linkage.

2. The conjugate of claim 1, wherein the analyte is a macromolecule selected from the group consisting of antibodies, antibody fragments, avidin, streptavidin, neutravidin, nucleic acids, receptors, binding protein, allergens, cells, viruses and synthetic polymers.

3. The conjugate of claim 1, wherein the analyte is a small molecule selected from the group consisting of therapeutic drugs, steroids, hormones, vitamins and small peptides.

4. The conjugate of claim 1, wherein the hydrophilic modifier is selected from the group consisting of hexaethylene glycol and spermine derivatives.

22. A process for performing a competitive assay comprising:

(a) exposing a sample suspected of containing a target analyte to the acridinium conjugate of claim 3 and to a corresponding binding partner for the target analyte;

(b) determining the extent to which the conjugate is competitively prevented and/or displaced by target analyte from the sample from forming a binding interaction with the corresponding binding partner;

(c) correlating the determination made in step (b) above with the presence or amount of target analyte in the sample.

23. A process for performing a sandwich assay comprising:

(a) exposing a sample suspected of containing a target analyte to the acridinium conjugate of claim 2 and to a corresponding binding partner for the target analyte, wherein said acridinium conjugate and said binding partner are capable of binding to different regions of the same target analyte to form a sandwich;

(b) initiating the chemiluminescent reaction to evolve detectable light from the sandwich;

(c) measuring the amount of light detected from the sandwich;

(d) correlating the amount of the detected light with the presence or amount of target analyte in the sample.

5. Acridinium ester conjugates of hydrophilic modifiers with the following structure:

wherein,

R₁ is alkyl, alkyenyl, alkynyl, aryl, sulfoethyl, sulfopropyl, sulfobutyl, or aralkyl having up to 24 carbons and up to 20 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorus and sulfur;

R₂, R₃, R₅, R₇ are hydrogen, amino, hydroxyl, halide, nitro, —

CN, SO₃H, —

SCN, —

OR, NHCOR, —

COR, —

COOR, or —

CONHR, wherein R is alkyl, alkenyl, alkynyl, aryl, aralkyl, having up to 24 carbons and up to 20 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorus and sulfur;

R₄ and R₈ are alkyl, alkenyl, alkynyl, aralkyl or alkoxyl having up to 8 carbons;

R₆═

R—

L—

S—

R₁₀, wherein R₆ has greater than 5 heteroatoms and is alternatively attached on a position of the phenoxy ring which is meta to the ester linkage such that R₅ or R₇ is attached para to the ester linkage;

R is optionally alkyl, alkenyl, alkynyl, aryl, aralkyl, having up to 24 carbons and up to 20 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorus and sulfur;

L is selected from the group consisting of ether, thioether, amide, ester and carbamate;

S is selected from the group consisting of polyethylene glycol varying from 280 to 5000 molecular weight and the following structures;

and R₁₀ is an electrophile, a leaving group, or a nucleophile.

6. The acridinium ester conjugate of claim 5, with the following structure:

7. The acridinium ester conjugate of claim 5, with the following structure:

8. The acridinium ester conjugate of claim 5, with the following structure:

9. The acridinium ester conjugate of claim 5, with the following structure:

10. A further acridinium ester conjugate comprising the acridinium ester conjugate of claim 5 and an analyte.

11. The further acridinium ester conjugate of claim 10, wherein the analyte is a small molecule selected from the group consisting of therapeutic drugs, steroids, hormones, vitamins and small peptides.

12. The further acridinium conjugate of claim 10, wherein the analyte is a macromolecule selected from the group consisting of antibodies, antibody fragments, avidin, streptavidin, neutravidin, nucleic acids, receptors, binding protein, allergens, cells, viruses and synthetic polymers.

13. The further acridinium ester conjugate of claim 11 wherein said small molecule is pteroic acid and said conjugate has the following structure:

wherein M is the acridinium ester.

14. The further acridinium ester conjugate of claim 13, having the following structure:

15. The further acridinium ester conjugate of claim 13, having the following structure:

16. The further acridinium ester conjugate of claim 13, having the following structure:

17. The further acridinium ester conjugate of claim 11, wherein said therapeutic drug is tobramycin and said conjugate has the following structure:

wherein M is the acridinium ester.

18. The further acridinium ester conjugate of claim 17 having the following structure:

19. The further acridinium ester of claim 11, where said therapeutic drug is theophylline and said conjugate has the following structure:

wherein M is the acridinium ester.

20. The further acridinium ester conjugate of claim 19 having the following structure:

21. The further acridinium ester conjugate of claim 19 having the following structure:

24. A method for improving the hydrophilic properties of a conjugate having the general structure A—

B—

C, wherein,

A is an analyte;

B is a hydrophilic modifier selected from the group consisting of polyethylene glycol having a molecular weight of about 280-5000, or a polyionic spacer derived from a polyamine where the internal, but not necessarily all the amines have been modified by hydrophilic molecules; and

C is a chemiluminescent acridinium compound containing a phenyl moiety with an ester linkage, comprising conjugating and introducing the hydrophilic modifier onto the phenyl moiety in a position meta or para to the ester linkage to improve the hydrophilic properties.

25. A method for improving the hydrophilic properties of acridinium ester conjugates with the following structure:

wherein

R₁ is alkyl, alkyenyl, alkynyl, aryl, sulfoethyl, sulfopropyl, sulfobutyl, or aralkyl having up to 24 carbons and up to 20 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorus and sulfur;

R₂, R₃, R₅, R₇ are hydrogen, amino, hydroxyl, halide, nitro, —

CN, SO₃H, —

SCN, —

OR, NHCOR, —

COR, —

COOR, or —

CONHR, wherein R is alkyl, alkenyl, alkynyl, aryl, aralkyl, having up to 24 carbons and up to 20 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorus and sulfur;

R₄ and R₈ are alkyl, alkenyl, alkynyl, aralkyl or alkoxyl having up to 8 carbons;

R₆═

R—

L—

S—

R₁₀, wherein R₆ has greater than 5 heteroatoms and is alternatively attached on a position of the phenoxy ring which is meta to the ester linkage such that R₅ or R₇ is attached para to the ester linkage;

R is optionally alkyl, alkenyl, alkynyl, aryl, aralkyl, having up to 24 carbons and up to 20 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorus and sulfur;

L is selected from the group consisting of ether, thioether, amide, ester and carbamate;

S is selected from the group consisting of polyethylene glycol varying from 280 to 5000 molecular weight and the following structures;

and R₁₀ is an electrophile, a leaving group, or a nucleophile, said method comprising conjugating and introducing S onto the phenoxy ring in a position meta or para to the ester linkage to improve the hydrophilic properties.