Chemiluminescent acridinium salts

• US 5,468,646 A
• Filed: 01/03/1995
• Issued: 11/21/1995
• Est. Priority Date: 10/22/1986
• Status: Expired due to Term

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Mattingly, Phillip G., Bennett, Larry G.
• Primary Examiner(s): Spiegel, Carol A.
• Application Number:
  US08/368,258
• Time in Patent Office: 322 Days
• Field of Search: 435/6, 435/7.1, 435/968, 436/501, 436/518, 436/536, 436/544, 436/546, 436/548, 436/800, 436/805, 530/389.8, 536/27.1, 546/104
• US Class Current: 436/501
• CPC Class Codes: C07D 219/16 with acyl radicals, substituted by nitrogen atoms, attached to the ring nitrogen atom
• CPC Class Codes: C07D 221/12 Phenanthridines
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C09K 11/07 having chemically interreactive components, e.g. reactive chemiluminescent compositions
• 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
• CPC Class Codes: Y10S 436/805 Optical property

Abstract

Acridinium sulfonylamides and isomers, such as phenanthridinium sulfonylamides, may be employed in applications including chemiluminmescent immunoassays. Methods for synthesis of these compounds include contacting an amine with a sulfonylhalide to form a sulfonamide and acylating with an activated carboxylic acid of an acridine or isomer thereof. The N-sulfonyl-9-acridinium carboxamide and isomers may be conjugated to antigens, haptens, antibodies, and nucleic acids for use in chemiluminescent assays.

Claims

1. A chemiluminescent conjugate comprising an antibody conjugated to a chemiluminescent compound of the formula:

##STR17## wherein R, R'"'"', R", X¹, X², and X³ independently comprise substituents which do not interfere with chemiluminescence measurement, linkage to the antibody is through one of X¹, X² and X³, and wherein Y-- is an anion with the proviso that R-X³, R'"'"'-X² and R"-X¹ may be hydrogen,

wherein X¹, X² and X³ are independently members of the group consisting of hydrogen, (--CO₂ H), (--C(═

O)OZ¹) wherein Z¹ is alkyl, (--C(═

O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═

O)OZ⁴) wherein Z⁴ is aryl, (--CN), (--C(═

O)N(Z⁵)--C(═

O)--Z⁵) wherein Z⁵ is H, alkyl or aryl, (F, Cl, Br, or I), (--N═

C═

O), (--N═

C═

S), (--SO₃^-), sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═

O)--Z⁷) wherein Z⁷ is (F, Cl, or Br), ##STR18## groups;

wherein R independently comprises a member selected from the group consisting of alkylene, alkenylene and substituted alkylene groups and wherein R'"'"' and R" independently comprise a member selected from the group consisting of alkylene, arylene, alkenylene, substituted alkylene, and substituted arylene groups, such that;

one or more hydrogens or open valences of said member is replaced by an alkyl, aryl, alkylene, alkenylene, substituted alkyl, substituted alkylene, substituted aryl, alkoxy, aryloxy, halo, nitro, amino, protected amino, substituted amino, hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substituted mercapto group;

or such that one or more carbon atoms of the member is replaced by a heteroatom.

2. In a method for performing a chemiluminescent assay to test for the presence of a member of a specific binding pair which may be present in a test sample wherein said method comprises (a) contacting said test sample suspected of containing said specific binding pair member with a conjugate comprising an antibody attached to a chemiluminescent compound capable of generating a detectable signal, which conjugate specifically reacts with said specific binding pair member and (b) determining the presence of said specific binding pair member by detecting the signal generated by said conjugate, wherein the improvement comprises the step of contacting said test sample suspected of containing said specific binding pair member with said chemiluminescent conjugate as recited in claim 92 wherein said antibody of said conjugate specifically binds said specific binding pair member.

3. A chemiluminescent conjugate comprising an antigen conjugated to a chemiluminescent compound of the formula:

##STR19## wherein R, R'"'"', R", X¹, X², and X³ independently comprise substituents which do not interfere with chemiluminescence measurement, linkage to the antigen is through one of X¹, X² and X³, and wherein Y-- is an anion with the proviso that R-X³, R'"'"'-X² and R"-X¹ may be hydrogen,

wherein X¹, X² and X³ are independently members of the group consisting of hydrogen, (--CO₂ H), (--C(═

O)OZ¹) wherein Z¹ is alkyl, (--C(═

O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═

O)OZ⁴) wherein Z⁴ is aryl, (--CN), (--C(═

O)N(Z⁵)--C(═

O)--Z⁵) wherein Z⁵ is H, alkyl or aryl, (F, Cl Br, or I), (--N═

C═

O), (--N═

C═

S), (--SO₃^-), sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═

O)--Z⁷) wherein Z⁷ is (F, Cl, or Br), ##STR20## groups;

wherein R independently comprises a member selected from the group consisting of alkylene, alkenylene and substituted alkylene groups and wherein R'"'"' and R" independently comprise a member selected from the group consisting of alkylene, arylene, alkenylene, substituted alkylene, and substituted arylene groups, such that;

one or more hydrogens or open valences of said member is replaced by an alkyl, aryl, alkylene, alkenylene, substituted alkyl, substituted alkylene, substituted aryl, alkoxy, aryloxy, halo, nitro, amino, protected amino, substituted amino, hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substituted mercapto group;

or such that one or more carbon atoms of the member is replaced by a heteroatom.

4. In a method for performing a chemiluminescent assay to test for the presence of a member of a specific binding pair which may be present in a test sample wherein said method comprises (a) contacting said test sample suspected of containing said specific binding pair member with a conjugate comprising an antigen attached to a chemiluminescent compound capable of generating a detectable signal, which conjugate specifically reacts with said specific binding pair member and (b) determining the presence of said specific binding pair member by detecting the signal generated by said conjugate, wherein the improvement comprises the step of contacting said test sample suspected of containing said specific binding pair member with said chemiluminescent conjugate as recited in claim 3 wherein said antigen of said conjugate specifically binds said specific binding pair member.

5. A chemiluminescent conjugate comprising a polynucleotide conjugated to a chemiluminescent compound of the formula:

##STR21## wherein R, R'"'"', R", X¹, X², and X³ independently comprise substituents which do not interfere with chemiluminescence measurement, linkage to the polynucleotide is through one of X¹, X² and X³, and wherein Y-- is an anion with the proviso that R-X³, R'"'"'-X² and R"-X¹ may be hydrogen,

wherein X¹, X² and X³ are independently members of the group consisting of hydrogen, (--CO₂ H), (--C(═

O)OZ¹) wherein Z¹ is alkyl, (--C(═

O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═

O)OZ⁴) wherein Z⁴ is aryl, (--CN), (--C(═

O)N(Z⁵)--C(═

O)--Z⁵) wherein Z⁵ is H, alkyl or aryl, (F, Cl Br, or I), (--N═

C═

O), (--N═

C═

S), (--SO₃^-), sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═

O)--Z⁷) wherein Z⁷ is (F, Cl, or Br), ##STR22## groups;

wherein R independently comprises a member selected from the group consisting of alkylene, alkenylene and substituted alkylene groups and wherein R'"'"' and R" independently comprise a member selected from the group consisting of alkylene, arylene, alkenylene, substituted alkylene, and substituted arylene groups, such that;

one or more hydrogens or open valences of said member is replaced by an alkyl, aryl, alkylene, alkenylene, substituted alkyl, substituted alkylene, substituted aryl, alkoxy, aryloxy, halo, nitro, amino, protected amino, substituted amino, hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substituted mercapto group;

or such that one or more carbon atoms of the member is replaced by a heteroatom.

6. In a method for performing a chemiluminescent assay to test for the presence of a polynucleotide which may be present in a test sample wherein said method comprises (a) contacting said test sample suspected of containing said polynucleotide with a conjugate comprising a polynucleotide complementary to said polynucleotide in said test sample which complementary polynucleotide specifically reacts with said polynucleotide of said test sample and which complementary polynucleotide is attached to a chemiluminescent compound capable of generating a detectable signal and (b) determining the presence of said polynucleotide by detecting the signal generated by said conjugate, wherein the improvement comprises the step of contacting said test sample suspected of containing said polynucleotide with said chemiluminescent conjugate as recited in claim 96 which polynucleotide of said conjugate is complementary to said polynucleotide in said test sample and wherein said complementary polynucleotide of said conjugate hybridizes with said polynucleotide in said test sample.

7. A chemiluminescent conjugate comprising a non-antibody, binding-protein conjugated to a chemiluminescent compound of the formula:

##STR23## wherein R, R'"'"', R", X¹, X², and X³ independently comprise substituents which do not interfere with chemiluminescence measurement, linkage to the non-antibody, binding-protein is through one of X¹, X² and X³, and wherein Y-- is an anion with the proviso that R-X³, R'"'"'-X² and R"-X¹ may be hydrogen,

wherein X¹, X² and X³ are independently members of the group consisting of hydrogen, (--CO₂ H), (--C(═

O)OZ¹) wherein Z¹ is alkyl, (--C(═

O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═

O)OZ⁴) wherein Z⁴ is aryl, (--CN), (--C(═

O)N(Z⁵)--C(═

O)--Z⁵) wherein Z⁵ is H, alkyl or aryl, (F, Cl Br, or I), (--N═

C═

O), (--N═

C═

S), (--SO₃^-), sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═

O)--Z⁷) wherein Z⁷ is (F, Cl, or Br), ##STR24## groups;

wherein R independently comprises a member selected from the group consisting of alkylene, alkenylene and substituted alkylene groups and wherein R'"'"' and R" independently comprise a member selected from the group consisting of alkylene, arylene, alkenylene, substituted alkylene, and substituted arylene groups, such that;

one or more hydrogens or open valences of said member is replaced by an alkyl, aryl, alkylene, alkenylene, substituted alkyl, substituted alkylene, substituted aryl, alkoxy, aryloxy, halo, nitro, amino, protected amino, substituted amino, hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substituted mercapto group;

or such that one or more carbon atoms of the member is replaced by a heteroatom.

8. In a method for performing a chemiluminescent assay to test for the presence of a complementary analyte of a non-antibody, binding-protein which may be present in a test sample wherein said method comprises (a) contacting said test sample suspected of containing said complementary analyte of said non-antibody, binding protein with a conjugate comprising a non-antibody, binding protein which specifically reacts with said complementary analyte of said non-antibody, binding protein and which non-antibody, binding-protein is attached to a chemiluminescent compound capable of generating a detectable signal and (b) determining the presence of said complementary analyte of said non-antibody, binding protein by detecting the signal generated by said conjugate, wherein the improvement comprises the step of contacting said test sample suspected of containing said complementary analyte with said chemiluminescent conjugate as recited in claim 7 wherein said non-antibody, binding-protein of said conjugate specifically binds said complementary analyte.

9. A chemiluminescent conjugate comprising a hapten conjugated to a chemiluminescent compound of the formula:

##STR25## wherein R, R'"'"', R", X¹, X², and X³ independently comprise substituents which do not interfere with chemiluminescence measurement, linkage to the hapten is through one of X¹, X² and X³, and wherein Y-- is an anion with the proviso that R-X³, R'"'"'-X² and R"-X¹ may be hydrogen,

wherein X¹, X² and X³ are independently members of the group consisting of hydrogen, (--CO₂ H), (--C(═

O)OZ¹) wherein Z¹ is alkyl, (--C(═

O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═

O)OZ⁴) wherein Z⁴ is aryl, (--CN), (--C(--O)N(Z⁵)--C(═

O)--Z⁵) wherein Z⁵ is H, alkyl or aryl, (F, Cl, Br, or I), (--N═

C═

O), (--N--C═

S), (--SO₃^-), sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═

O)--Z⁷) wherein Z⁷ is (F, Cl, or Br), ##STR26## groups;

wherein R independently comprises a member selected from the group consisting of alkylene, alkenylene and substituted alkylene groups and wherein R'"'"' and R" independently comprise a member selected from the group consisting of alkylene, arylene, alkenylene, substituted alkylene, and substituted arylene groups, such that;

one or more hydrogens or open valences of said member is replaced by an alkyl, aryl, alkylene, alkenylene, substituted alkyl, substituted alkylene, substituted aryl, alkoxy, aryloxy, halo, nitro, amino, protected amino, substituted amino, hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substituted mercapto group;

or such that one or more carbon atoms of the member is replaced by a heteroatom.

10. In a method for performing a competitive chemiluminescent assay to test for the presence of a hapten which may be present in a test sample wherein said method comprises (a) contacting said test sample suspected of containing said hapten with a known amount of an anti-hapten antibody specific for said hapten and with a conjugate comprising a hapten which competes with said hapten in said test sample for binding with said anti-hapten antibody and wherein said hapten of said conjugate is attached to a chemiluminescent compound capable of generating a detectable signal and (b) determining the presence of said hapten in said test sample by detecting the signal generated by said conjugate, wherein the improvement comprises the step of contacting said test sample suspected of containing said hapten with said chemiluminescent conjugate as recited in claim 9 wherein said hapten of said conjugate competes with said hapten in said test sample for specific binding to said anti-hapten antibody.

11. A chemiluminescent conjugate comprising a small molecular weight analyte which binds to a binding protein conjugated to a chemiluminescent compound of the formula:

##STR27## wherein R, R'"'"', R", X¹, X², and X³ independently comprise substituents which do not interfere with chemiluminescence measurement, linkage to the small molecular weight analyte is through one of X¹, X² and X³, and wherein Y-- is an anion with the proviso that R-X³, R'"'"'-X² and R"-X¹ may be hydrogen,

wherein X¹, X² and X³ are independently members of the group consisting of hydrogen, (--CO₂ H), (--C(═

O)OZ¹) wherein Z¹ is alkyl, (--C(═

O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═

O)OZ⁴) wherein Z4 is aryl, (--CN), (--C(═

O)N(Z⁵)--C(--O)--Z⁵) wherein Z⁵ is H, alkyl or aryl, (F, Cl, Br, or I), (--N═

C═

O), (--N═

C═

S), (--SO₃^-), sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═

O)--Z⁷) wherein Z⁷ is (F, Cl, or Br), ##STR28## groups;

wherein R independently comprises a member selected from the group consisting of alkylene, alkenylene and substituted alkylene groups and wherein R'"'"' and R" independently comprise a member selected from the group consisting of alkylene, arylene, alkenylene, substituted alkylene, and substituted arylene groups, such that;

one or more hydrogens or open valences of said member is replaced by an alkyl, aryl, alkylene, alkenylene, substituted alkyl, substituted alkylene, substituted aryl, alkoxy, aryloxy, halo, nitro, amino, protected amino, substituted amino, hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substituted mercapto group;

or such that one or more carbon atoms of the member is replaced by a heteroatom.

12. In a method for performing a competitive chemiluminescent assay to test for the presence of a small molecular weight analyte which binds to a binding protein and which may be present in a test sample wherein said method comprises (a) contacting said test sample suspected of containing said small molecular weight analyte with a known amount of said binding protein specific for said small molecular weight analyte and with a conjugate comprising a small molecular weight analyte which competes with said small molecular weight analyte in said test sample and which binds to said binding protein, wherein said small molecular weight analyte of said conjugate is attached to a chemiluminescent compound capable of generating a detectable signal and (b) determining the presence of said small molecular weight analyte by detecting the signal generated by said conjugate, wherein the improvement comprises the step of contacting said test sample suspected of containing said small molecular weight analyte with said chemiluminescent conjugate as recited in claim 11 wherein said small molecular weight analyte of said conjugate competes with said small molecular weight analyte in said test sample for specific binding to said binding protein.

13. A chemiluminescent conjugate formed by a member selected from the group consisting of an antigen, an antibody, a hapten, a polynucleotide, a small molecular weight analyte which binds to a binding protein and a non-antibody, binding-protein conjugated to a chemiluminescent compound of the formula:

##STR29## wherein R independently comprises a member selected from the group consisting of alkylene, alkenylene and substituted alkylene groups such that;

one or more hydrogens or open valences of said member is replaced by an alkyl, aryl, alkylene, alkenylene, substituted alkyl, substituted alkylene, substituted aryl, alkoxy, aryloxy, halo, nitro, amino, protected amino, substituted amino, hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substituted mercapto group;

or such that one or more carbon atoms of said member is replaced by a heteroatom;

Y-- is an anion;

wherein conjugation is through X³ and wherein X³ is independently a member of the group consisting of hydrogen, (--CO₂ H), (--C(═

O)OZ¹) wherein Z¹ is alkyl, (--C(═

O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═

O)OZ⁴) wherein Z⁴ is aryl, (--CN), (--C(═

O)N(Z⁵)--C(═

O)--Z⁵) wherein Z⁵ is H, alkyl or aryl, (F, Cl, Br, or I), (--N═

C═

O), (--N═

C═

S), (--SO₃^-), sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═

O)--Z⁷) wherein Z⁷ is (F, Cl, or Br), ##STR30## groups; and

with the proviso that R-X³ may also be hydrogen.

14. The chemiluminescent conjugate as recited in claim 13 wherein said heteroatom is selected from the group consisting of nitrogen, phosphorus, sulfur and oxygen.

15. The chemiluminescent conjugate as recited in claim 13 wherein R independently is of the formula --(CH₂)_n --, where n=0-50.

18. The method as recited in claim 13 wherein R of said chemiluminescent conjugate independently is of the formula --(CH₂)_n --, where n=0-50.

16. A method for performing a chemiluminescent immunoassay to test for the presence of an analyte which may be present in a test sample comprising:

(a) contacting said test sample suspected of containing said analyte with a chemiluminescent conjugate that specifically binds said analyte and which conjugate comprises a member selected from the group consisting of an antigen, an antibody, a hapten, a polynucleotide, a small molecular weight analyte which binds to a binding protein and a non-antibody, binding-protein conjugated through X³ of a chemiluminescent compound of the formula;

##STR31## wherein R independently comprises a member selected from the group consisting of alkylene, alkenylene and substituted alkylene groups such that;

one or more hydrogens or open valences of said member is replaced by an alkyl, aryl, alkylene, alkenylene, substituted alkyl, substituted alkylene, substituted awl, alkoxy, aryloxy, halo, nitro, amino, protected amino, substituted amino, hydroxy, protected hydroxy, oxo, thio, imino, mercapto or substituted mercapto group;

or such that one or more carbon atoms of said member is replaced by a heteroatom;

Y-- is an anion;

wherein X³ is independently a member of the group consisting of hydrogen, (--CO₂ H), (--C(═

O)OZ¹) wherein Z¹ is alkyl, (--C(═

O)NZ² Z³) wherein Z² or Z³ is H, alkyl or aryl, (--C(═

O)OZ⁴) wherein Z⁴ is aryl, (--CN), (--C(═

O)N(Z⁵)--C(═

O)--Z⁵) wherein Z⁵ is H, alkyl or aryl, (F, Cl, Br, or I), (--N═

C═

O), (--N═

C═

S), (--SO₃^-), sulfonyl halide (--SO₂ Z⁶) wherein Z⁶ is (Cl or F), (--C(═

O)--Z⁷) wherein Z⁷ is (F, Cl, or Br), ##STR32## groups; and

with the proviso that R-X³ may also be hydrogen; and

(b) detecting a signal generated from said chemiluminescent conjugate as an indication of the presence of said analyte in said test sample.

17. The method as recited in claim 16 wherein said heteroatom of said chemiluminescent conjugate is selected from the group consisting of nitrogen, phosphorus, sulfur and oxygen.

19. A chemiluminescent conjugate comprising a member selected from the group consisting of an antigen, an antibody, a hapten, a polynucleotide, a small molecular weight analyte which binds to a binding protein and a non-antibody, binding-protein conjugated through either the CO₂ H or SO₃^- group of a chemiluminescent compound of the formula:

##STR33## wherein Y-- is an anion.

20. A method for performing a chemiluminescent immunoassay to test for the presence of an analyte which may be present in a test sample comprising:

(a) contacting said test sample suspected of containing said analyte with a chemiluminescent conjugate that specifically binds said analyte and which conjugate comprises a member selected from the group consisting of an antigen, an antibody, a hapten, a polynucleotide, a small molecular weight analyte which binds to a binding protein and a non-antibody, binding-protein conjugated through either the CO₂ H or SO₃^- group of a chemiluminescent compound of the formula;

##STR34## wherein Y-- is an anion; and

(b) detecting a signal generated from said chemiluminescent conjugate as an indication of the presence of said analyte in said test sample.

21. A chemiluminescent conjugate comprising a member selected from the group consisting of an antigen, an antibody, a hapten, a polynucleotide, a small molecular weight analyte which binds to a binding protein and a non-antibody, binding-protein conjugated through either SO₃^- group of a chemiluminescent compound of the formula:

##STR35## wherein Y-- is an anion.

22. A method for performing a chemiluminescent immunoassay to test for the presence of an analyte which may be present in a test sample comprising:

(a) contacting said test sample suspected of containing said analyte with a chemiluminescent conjugate that specifically binds said analyte and which conjugate comprises a member selected from the group consisting of an antigen, an antibody, a hapten, a polynucleotide, a small molecular weight analyte which binds to a binding protein and a non-antibody, binding-protein conjugated through either SO₃^- group of a chemiluminescent compound of the formula;

##STR36## wherein Y-- is an anion; and

(b) detecting a signal generated from said chemiluminescent conjugate as an indication of the presence of said analyte in said test sample.

23. A chemiluminescent conjugate comprising a member selected from the group consisting of an antigen, an antibody, a hapten, a polynucleotide, a small molecular weight analyte which binds to a binding protein and a non-antibody, binding-protein conjugated through the CO₂ H group of a chemiluminescent compound of the formula selected from the group consisting of:

##STR37## wherein Y-- is an anion.

24. A method for performing a chemiluminescent immunoassay to test for the presence of an analyte which may be present in a test sample comprising:

(a) contacting said test sample suspected of containing said analyte with a chemiluminescent conjugate that specifically binds said analyte and which conjugate comprises a member selected from the group consisting of an antigen, an antibody, a hapten, a polynucleotide, a small molecular weight analyte which binds to a binding protein and a non-antibody, binding-protein conjugated through the CO₂ H group of a chemiluminescent compound of the formula selected from the group consisting of;

##STR38## wherein Y-- is an anion; and

(b) detecting a signal generated from said chemiluminescent conjugate as an indication of the presence of said analyte in said test sample.

25. A chemiluminescent conjugate comprising a member selected from the group consisting of an antigen, an antibody, a hapten, a polynucleotide, a small molecular weight analyte which binds to a binding protein and a non-antibody, binding-protein conjugated through either the CO₂ H or SO₃ -- group of a chemiluminescent compound of the formula selected from the group consisting of:

##STR39## wherein Y-- is an anion.

26. A method for performing a chemiluminescent immunoassay to test for the presence of an analyte which may be present in a test sample comprising:

(a) contacting said test sample suspected of containing said analyte with a chemiluminescent conjugate that specifically binds said analyte and which conjugate comprises a member selected from the group consisting of an antigen, an antibody, a hapten, a polynucleotide, a small molecular weight analyte which binds to a binding protein and a non-antibody, binding-protein conjugated through either the CO₂ H or SO₃ -group of a chemiluminescent compound of the formula selected from the group consisting of;

##STR40## wherein Y-- is an anion; and

(b) detecting a signal generated from said chemiluminescent conjugate as an indication of the presence of said analyte in said test sample.