Mixed luminescent conjugate test

• US 5,672,475 A
• Filed: 01/20/1995
• Issued: 09/30/1997
• Est. Priority Date: 05/06/1993
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Chiron Corporation (Bayer AG)
• Inventors: Lee, Michael J., Weetall, Howard H., Connolly, Joseph E.
• Primary Examiner(s): Green, Lora M.
• Application Number:
  US08/375,466
• Time in Patent Office: 984 Days
• Field of Search: 435/6, 435/7.72, 435/7.7, 435/28, 435/967, 435/968, 435/973, 435/975, 436/518, 436/523, 436/526, 436/528, 436/172, 436/805
• US Class Current: 435/6.11
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte

Abstract

A method is provided for detecting or quantitating each of a plurality of substances or at least one substance and an internal reference material or control material in a test sample using at least two different luminescent labelled conjugates. Each luminescent labelled conjugate being characterized in being activated to emit light under different process conditions. The invention also provides for test kits containing at least two different luminescent labelled conjugates for detecting or quantitating presence or absence of at least two substances or at least one substance an internal reference material or control material in a test sample.

Claims

1. A dual luminescent label specific binding assay for detecting two substances in a sample comprising the sequential steps of:

(a) forming a test reaction mixture comprising;

(i) a test sample comprising a first substance and a second substance;

(ii) a first test reagent comprising a first binding partner (BP1) which specifically binds to said first substance wherein said first binding partner is attached to a first luminescent label selected from the group consisting of luminols and isoluminols (BP1_L) and a second binding partner (BP2) which is immobilized on a solid support (BP2_S); and

(iii) a second test reagent comprising a third binding partner (BP3) which specifically binds to said second substance wherein said third binding partner is attached to a second luminescent label selected from the group consisting of acridinium esters and benzacridinium esters (BP3_L) and a fourth binding partner (BP4) which specifically binds to said second substance, wherein said fourth binding partner is immobilized on a solid support (BP4_S);

(b) reacting said test mixture to form a first specific binding complex comprising BP1_L -first substance-BP2_S, and a second specific binding complex comprising BP3_L -second substance- BP4_S ;

(c) separating said first and second binding complexes from unbound BP1_L and BP3_L ;

(d) activating said first luminescent label in the presence of an oxidation catalyst at a first pH process condition to emit a first measurable light emission from said first luminescent label, wherein said first pH process condition does not activate said second luminescent label;

(e) measuring said first light emission;

(f) activating said second luminescent label at a second pH process condition to emit a second measurable light emission from said second luminescent label;

(g) measuring said second light emission; and

(h) correlating the first light emission to the presence or amount of said first substance and correlating the second light emission to the presence or amount of said second substance present in said test sample,

wherein said first pH condition is a pH level lower than said second pH condition.

2. A method according to claim 1 wherein said first and second binding partners are antibodies which bind to said first substance, wherein said first substance is an antigen, and said third and fourth binding partners are antibodies which bind to said second substance, wherein said second substance is an antigen.

3. A method according to claim 2 wherein said first luminescent label is selected from the group consisting of N-(4-aminobutyl)-N-ethylisoluminol and 5-amino-2,3-dihydro-1,4-phthalazine dione and said second luminescent label is dimethyl acridinium ester.

4. A method according to claim 1 wherein said first pH condition is 7.4.

5. A method according to claim 1 wherein said first label is N-(4-aminobutyl)-N-ethylisoluminol and said second label is a dimethyl acridinium ester and said second label is activated upon the sequential addition of a first solution comprising hydrogen peroxide and nitric acid followed by a second solution comprising sodium hydroxide.

6. A method according to claim 5 wherein said first label is activated with an activating agent comprising hydrogen peroxide in the presence of a microperoxidase catalyst.

7. A dual luminescent label specific binding assay for detecting two substances in a sample comprising the sequential steps of:

(a) forming a test reaction mixture comprising;

(i) a test sample comprising a first substance and a second substance;

(ii) a first test reagent comprising a first binding partner (BP1) which of specifically binds to said first substance and a first competitor (C1) which competes with said first substance for binding to said first binding partner, wherein said first binding partner is immobilized on a solid support (BP1_S) and said first competitor to said first substance is attached to a first luminescent label selected from the group consisting of luminols and isoluminols (C1_L);

(iii) a second test reagent comprising a second binding partner (BP2) which specifically binds to said second substance and a second competitor (C2) which competes with said second substance for binding to said second binding partner wherein said second binding partner is immobilized on a solid support (BP2_S) and said second competitor to said second substance is attached to a second luminescent label selected from the group consisting of acridinium esters and benzacridinium esters (C2_L),

(b) reacting said test mixture to form a first specific binding complex comprising either BP1_S -first substance or BP1_S -C1_L, and a second specific binding complex comprising either BP2_S -second substance or BP2_S -C2_L ;

(c) separating said first and second complexes from said unbound C1_L and C2_L ;

(d) activating said first luminescent label in the presence of an oxidation catalyst at a first pH process condition to emit a first measurable light emission wherein said first pH process condition does not activate said second luminescent label;

(e) measuring said first light emission;

(f) activating said second luminescent label at a second pH process condition to emit a second measurable light emission;

(g) measuring said second light emission; and

(h) correlating the first light emission to the presence or amount of said first substance and correlating the second light emission to the presence or amount of said second substance present in said test sample,

wherein said first pH condition is a pH level lower than said second pH condition.

8. A method according to claim 7 wherein said first luminescent label is selected from the group consisting of N-(4-aminobutyl)-N-ethylisoluminol and 5-amino-2,3-dihydro-1,4-phthalazine dione.

9. A method as recited in claim 8, wherein said first and second substances are selected from the group consisting of antigens, antibodies, haptens, hormones, receptors, nucleic acids, nucleic acid probes, toxins, organic chemicals, drugs, infectious agents, internal reference materials and control materials.

10. A method as recited in claim 9, wherein said first label is activated at a pH condition of 7.4.

11. A method as recited in claim 8, wherein said first luminescent label is N-(4-aminobutyl)-N-ethylisoluminmol and said second label is dimethyl acridinium ester, wherein said first label is activated by addition of a microperoxidase oxidation catalyst in the presence of a hydrogen peroxide solution and said second label is activated upon the sequential addition of a first solution comprising hydrogen peroxide and a nitric acid followed by a second solution comprising sodium hydroxide.

12. A dual luminescent label specific binding assay for detecting a substance and an internal reference material comprising the sequential steps of:

(a) forming a test reaction mixture comprising;

(i) a test sample comprising a substance to be detected or quantitated;

(ii) a first test reagent comprising a binding partner which specifically binds to said substance and a competitor which competes with said substance for binding to said binding partner wherein said binding partner is immobilized on a solid support (BP_S) and said competitor is attached to a first luminescent label selected from the group consisting of luminols and isoluminols (C_L); and

(iii) a second test reagent comprising an internal reference material corresponding to said substance, wherein said internal reference material is attached to a second luminescent label selected from the group consisting of acridinium esters and benzacridinium esters (C_I),

(b) reacting said test mixture to form a specific binding complex comprising either BP_S -substance, BP_S -C_L or BP_S -C_I ;

(c) separating said complex from unbound C_L and C_I,

(d) activating said first luminescent label in the presence of an oxidation catalyst at a first pH process condition to emit a first measurable light emission wherein said first pH process condition does not activate said second luminescent label;

(e) measuring said first light emission;

(f) activating said second luminescent label at a second pH process condition to emit a second measurable light emission;

(g) measuring said second light emission; and

(h) correlating the first light emission to the presence or amount of said substance present in said test sample and correlating the second light emission to the presence or amount of said internal reference material,

wherein said first pH condition is a pH level lower than said second pH condition.

13. A method according to claim 12 wherein said first luminescent label is selected from the group consisting of N-(4-aminobutyl)-N-ethylisoluminol and 5-amino-2,3-dihydro-1,4-phthalazine dione and said second luminescent label is dimethyl acridinium ester.

14. A method according to claim 12 wherein said first pH condition is 7.4.

15. A method according to claim 12 wherein said second label is activated upon the sequential addition of a first solution comprising hydrogen peroxide and nitric acid followed by a second solution comprising sodium hydroxide.

16. A method according to claim 15 wherein said first label is activated with an activating agent comprising hydrogen peroxide in the presence of a microperoxidase catalyst and said first label is N-(4-aminobutyl)-N-ethylisoluminol.

17. A dual luminescent label specific binding assay for detecting a substance in a test sample and an internal reference material comprising the steps of:

(a) forming a test reaction mixture comprising;

(i) a test sample comprising a substance to be detected or quantitated;

(ii) a first test reagent comprising first and second binding partners which specifically bind to said substance, wherein said first binding partner is attached to a first luminescent label selected from the group consisting of luminols and isoluminols (BP1_L) and said second binding partner is immobilized on a solid support (BP2_S); and

(iii) a second test reagent comprising an internal reference material corresponding to said substance, wherein said internal reference material is attached to a second luminescent label selected from the group consisting of acridinium esters and benzacridinium esters (C_L);

(b) reacting said test mixture to form a specific binding complex comprising either BP2_S -substance-BP1_L or BP2_S -C_I -BP1_L ;

(c) separating said complex from unbound BP1_L and unbound C_I ;

(d) activating said first luminescent label in the presence of an oxidation catalyst at a first pH process condition to emit a first measurable light emission from said first luminescent label, wherein said first pH process condition does not activate said second luminescent label;

(e) measuring said first light emission;

(f) activating said second luminescent label at a second pH process condition to emit a second measurable light emission from said second luminescent label;

(g) measuring said second light emission; and

(h) correlating the first light emission to the presence or amount of said substance present in said test sample and correlating the second light emission to the presence or amount of said internal reference material,

wherein said first pH condition is a pH level lower than said second pH condition.

18. A method according to claim 17 wherein said assay is a hybridization assay said first luminescent label is selected from the group consisting of N-(4-aminobutyl)-N-ethylisoluminol and 5-amino-2,3-dihydro-1,4-phthalazine and said second luminescent label is dimethyl acridinium ester.

19. A method according to claim 17 wherein said first pH condition is 7.4.

20. A method according to claim 17 wherein said second label is activated upon the sequential addition of a first solution comprising hydrogen peroxide and nitric acid followed by a second solution comprising sodium hydroxide.

21. A method according to claim 20 wherein said first label is activated with an activating agent comprising hydrogen peroxide in the presence of a microperoxidase catalyst and said first label is N-(4-aminobutyl)-N-ethylisoluminol.

22. A dual luminescent label assay for detecting or measuring two substances in a sample comprising the sequential steps of:

(a) forming a test reaction mixture comprising;

(i) a test sample comprising a first substance and a second substance;

(ii) a first test reagent comprising a first binding partner (BP1) which specifically binds to said first substance wherein said first binding partner is immobilized on a solid support (BP1_S) and a second specific binding partner (BP2) which specifically binds to said first substance, wherein said second binding partner is attached to a first luminescent label selected from the group consisting of luminols and isoluminols (BP2_L); and

(iii) a second test reagent comprising a third binding partner (BP3) which specifically binds to said second substance and a competitor which competes with said second substance for binding to said third binding partner, wherein said third binding partner is immobilized on a solid support (BP3_S) and said competitor is attached to a second luminescent label selected from the group consisting of acridinium esters and benzacridinium esters (C_L),

(b) reacting said test mixture to form a first specific binding complex comprising BP1_S -first substance-BP2_L, and a second specific binding complex comprising either BP3_S -second substance or BP3_S -C_L ;

(c) separating said first complex from unbound BP2_L and separating said second complex from unbound C_L ;

(d) activating said first luminescent label in the presence of an oxidation catalyst at a first pH process condition to emit a first measurable light emission from said first luminescent label, wherein said first pH process condition does not activate said second luminescent label;

(e) measuring said first light emission;

(f) activating said second luminescent label at a second pH process condition to emit a second measurable light emission;

(g) measuring said second light emission; and

(h) correlating the first light emission to the presence or amount of said first substance and correlating the second light emission to the presence or amount of said second substance present in said test sample,

wherein said first pH condition is a pH level lower than said second pH condition.

23. A method as recited in claim 22 wherein said first luminescent label is selected from the group consisting of N-(4-aminobutyl)-N-ethylisoluminol and 5-amino-2,3-dihydro-1,4-phthalazine dione and said second luminescent label is dimethyl acridinium ester.

24. A method according to claim 22 wherein said first pH condition is 7.4.

25. A method according to claim 22 wherein said second label is activated upon the sequential addition of a first solution comprising hydrogen peroxide and nitric acid followed by a second solution comprising sodium hydroxide and said first label is N-(4-aminobutyl)-N-ethylisoluminol.

26. A method according to claim 25 wherein said first label is activated with an activating agent comprising hydrogen peroxide in the presence of a microperoxidase catalyst.

27. A dual luminescent label specific binding assay for detecting or measuring a substance and an internal reference material comprising the sequential steps of:

(a) forming a test reaction mixture comprising;

(i) a test sample comprising a substance to be detected or quantitated;

(ii) a first test reagent comprising an internal reference material corresponding to said substance, wherein said internal reference material is attached to a first luminescent label selected from the group consisting of luminols and isoluminols (C_I); and

(iii) a second test reagent comprising a specific binding partner which specifically binds to said substance and a competitor which competes with said substance, wherein said binding partner is immobilized on a solid support (BP_S) and said competitor is attached to a second luminescent label selected from the group consisting of acridinium esters and benzacridinium esters (C_L),

(b) reacting said test mixture to form a first specific binding complex comprising BP_S -substance, BP_S -C_L or BP_S -C_I ;

(c) separating said complex from said unbound C_L and unbound C_I ;

(d) activating said first luminescent label in the presence of an oxidation catalyst at a first pH process condition to emit a first measurable light emission from said first luminescent label, wherein said first pH process condition does not activate said second luminescent label;

(e) measuring said first light emission;

(f) activating said second luminescent label at a second pH process condition to emit a second measurable light emission;

(g) measuring said second light emission; and

(h) correlating the first light emission to the presence or amount of said internal reference material and correlating the second light emission to the presence or amount of said substance present in said test sample,

wherein said first pH condition is a pH level lower than said second pH condition.

28. A method according to claim 27 wherein said first luminescent label is selected from the group consisting of N-(4-aminobutyl)-N-ethylisoluminol and 5-amino-2,3-dihydro-1,4-phthalazine dione and said second luminescent label is dimethyl acridinium ester.

29. A method according to claim 28 wherein said first pH condition is 7.4.

30. A method according to claim 27 wherein said second label is activated upon the sequential addition of a first solution comprising hydrogen peroxide and nitric acid followed by a second solution comprising sodium hydroxide and said first label is N-(4-aminobutyl)-N-ethylisoluminol.

31. A method according to claim 30 wherein said first label is activated with an activating agent comprising hydrogen peroxide in the presence of a microperoxidase catalyst.

32. A dual luminescent label specific binding assay for detecting or measuring a substance and an internal reference material comprising the sequential steps of:

(a) forming a test reaction mixture comprising;

(i) a test sample comprising a substance to be detected or quantitated;

(ii) a first test reagent comprising an internal reference material corresponding to said substance, wherein said internal reference material has attached thereto a first luminescent label selected from the group consisting of luminols and isoluminols (C_L); and

(iii) a second test reagent comprising first and second specific binding partners which specifically bind to said substance, wherein said first binding partner is immobilized on a solid support (BP1_S) and said second binding partner is attached to a second luminescent label selected from the group consisting of acridinium esters and benzacridinium esters (BP2_L),

(b) reacting said test mixture to form a specific binding complex comprising either BP1_S -substance-BP2_L or BP1_S -C_I -B2P_L ;

(c) separating said complexes from unbound C_I and BP2_L ;

(d) activating said first luminescent label in the presence of an oxidation catalyst at a first pH process condition to emit a first measurable light emission from said first luminescent label, wherein said first pH process condition does not activate said second luminescent label;

(e) measuring said first light emission;

(f) activating said second luminescent label at a second pH process condition to emit a second measurable light emission;

(g) measuring said second light emission; and

(h) correlating the first light emission to the presence or amount of said internal reference material and correlating the second light emission to the presence or amount of said substance present in said test sample,

wherein said first pH condition is a pH level lower than said second pH condition.

33. A method according to claim 32 wherein said first pH condition is 7.4 and said first luminescent label is selected from the group consisting of N-(4-aminobutyl)-N-ethylisoluminol and 5-amino-2,3-dihydro-1,4-phthalazine and said second luminescent label is dimethyl acridinium ester.

34. A method according to claim 32 wherein said second label is activated upon the sequential addition of a first solution comprising hydrogen peroxide and nitric acid followed by a second solution comprising sodium hydroxide.

35. A method according to claim 34 wherein said first label is activated with an activating agent comprising hydrogen peroxide in the presence of a microperoxidase catalyst and said first label is N-(4-aminobutyl)-N-ethylisoluminol.

36. A dual luminescent label specific binding assay for detecting two substances in a sample comprising the sequential steps of:

(a) forming a test reaction mixture comprising;

(i) a test sample comprising a first substance and a second substance to be detected or quantitated;

(ii) a first test reagent comprising a first binding partner which specifically binds to said first substance and a competitor which competes with said first substance for binding to said first binding partner wherein said first binding partner is immobilized on a solid support (BP1_S), and wherein said competitor to said first substance is attached to a first luminescent label selected from the group consisting of luminols and isoluminols (C_L); and

(iii) a second test reagent comprising a second binding partner (BP2) which specifically binds to said second substance, wherein said second binding partner is attached to a second luminescent label selected from the group consisting of acridinium esters and benzacridinium esters (BP2_L), and a third binding partner (BP3) which specifically binds to said second substance, wherein said third binding partner is immobilized on a solid support (BP3_S),

(b) reacting said test mixture to form a first specific binding complex comprising BP1_S -first substance or BP1_S -C_L, and a second complex comprising BP2_L -second substance-BP3_S ;

(c) separating said complexes from unbound BP2_L and unbound C_L ;

(d) activating said first luminescent label in the presence of an oxidation catalyst at a first pH process condition to emit a first measurable light emission from said first luminescent label, wherein said first pH process condition does not activate said second luminescent label;

(e) measuring said first light emission;

(f) activating said second luminescent label at a second pH process condition to emit a second measurable light emission from said second luminescent label;

(g) measuring said second light emission; and

(h) correlating the first light emission to the presence or amount of said first substance and correlating the second light emission to the presence or amount of said second substance present in said test sample,

wherein said first pH condition is a pH level lower than said second pH condition.

37. A method according to claim 36 wherein said second label is activated upon the sequential addition of a first solution comprising hydrogen peroxide and nitric acid followed by a second solution comprising sodium hydroxide and said first luminescent label is selected from the group consisting of N-(4-aminobutyl)-N-ethylisoluminol and 5-amino-2,3-dihydro-1,4-phthalazine and said second luminescent label is dimethyl acridinium ester.

38. A method according to claim 36 wherein said first label is activated with an activating agent comprising hydrogen peroxide in the presence of a microperoxidase catalyst and said first label is N-(4-aminobutyl)-N-ethylisoluminol.