Nucleic acid hybridization assay employing antibodies to intercalation complexes

• US 4,563,417 A
• Filed: 12/24/1984
• Issued: 01/07/1986
• Est. Priority Date: 08/31/1984
• Status: Expired due to Term

• Current Assignee: Miles Laboratories, Inc. (Bayer AG)
• Original Assignee: Miles Laboratories, Inc. (Bayer AG)
• Inventors: Albarella, James P., Anderson, Leslie H. D.
• Primary Examiner(s): Nucker, Christine M.
• Application Number:
  US06/685,903
• Time in Patent Office: 379 Days
• Field of Search: 435/6, 435/7, 436/504, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/705 for herpetoviridae, e.g. herpes simplex, varicella zoster
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: Y10S 435/966 involving an enzyme system with high turnover rate or complement magnified assay, e.g. multi-enzyme systems
• CPC Class Codes: Y10S 435/975 Kit

Abstract

Nucleic acid hybridization assay methods and reagent systems for detecting a particular polynucleotide sequence in a test medium. An aggregate is formed in the assay reaction mixture comprising intercalation complexes between a nucleic acid intercalator and double stranded nucleic acid associated with the hybridization product of the sequence to be detected and a nucleic acid probe sequence. Hybridization of the probe with the sequence to be detected can then be determined by addition of an antibody, or a fragment thereof, capable of binding with the intercalation complexes in the formed aggregate and measuring the antibody or fragment thereof which becomes bound to such intercalation complexes associated with hybridized probe. In one preferred embodiment, this method eliminates the need to chemically modify the probe in order to form a labeled reagent. In another embodiment, the method provides an advantageous method for labeling the probe by chemical modification.

Claims

1. A method for detecting a particular polynucleotide sequence in a test medium containing single stranded nucleic acids, comprising the steps of:

(a) combining the test medium with (i) a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to hybridization between the sequence to be detected and the complementary sequence in the probe, and (ii) a nucleic acid intercalator capable of binding to double stranded nucleic acid in the form of intercalation complexes, and

(b) detecting hydridized probe by adding an antibody, or a fragment thereof, capable of binding with intercalation complexes in the hybridization product resulting from step (a), and determining the antibody or fragment thereof which becomes bound to such complexes.

2. The method of claim 1 wherein the intercalator is combined with the test medium as a separate, free compound and noncovalently binds with double stranded nucleic acid to form intercalation complexes.

3. The method of claim 1 wherein the intercalator is chemically linked to the probe in the single stranded complementary region of the probe, whereby upon hybridization said intercalation complexes are formed in such region.

4. The method of claim 1 wherein the antibody or fragment thereof is labeled with a detectable chemical group.

5. The method of claim 4 wherein the detectable chemical group is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.

6. The method of claim 1 according to a solid phase hybridization technique wherein one of the probe and the single stranded nucleic acids from the test medium is immobilized on a solid support and wherein the antibody associated with the solid support is determined.

7. The method of claim 1 according to a solid phase sandwich hybridization technique wherein the test medium is combined with first and second nucleic acid probes each comprising at least one single stranded base sequence which is substantially complementary to a mutually exclusive portion of the sequence to be detected and wherein one of the probes is immobilized on a solid support.

8. The method of claim 1 according to a solution phase hybridization technique wherein the probe comprises a binding site for a binding substance and wherein after the hybridization step there is added an immobilized form of such binding substance.

9. The method of claim 8 wherein the probe comprises a biotin or hapten moiety and the binding substance is avidin or an anti-hapten antibody, respectively.

10. The method of claim 1 wherein the probe additionally comprises a double stranded portion which, upon addition of the intercalator in step(a) as a separate, free compound, forms said intercalation complexes.

11. The method of claim 1 wherein the intercalator is selected from acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.

12. A solid-phase hybridization method for detecting a particular polynucleotide sequence in a liquid test medium containing single stranded nucleic acids, comprising the steps of:

(a) forming a reaction mixture by contacting the liquid test medium with a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be detected, one of the probe and the single stranded nucleic acids from the test medium being immobilized on a solid support, such contact being performed under conditions favorable to hybridization between the sequence to be detected and the complementary probe sequence,

(b) contacting the solid support carrying resulting immobilized duplexes with a nucleic acid intercalator and an antibody, or fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator,

(c) separating the solid support carrying resulting immobilized antibody or fragment thereof from the remainder of the reaction mixture, and

(d) determining the separated antibody or fragment thereof on the solid support as an indication of the presence of the sequence to be detected in the liquid test medium.

13. The method of claim 12 wherein prior to step (b) the solid support carrying immobilized duplexes resulting from step(a) is separated from the remainder of the reaction mixture.

14. The method of claim 12 wherein the antibody or fragment thereof is labeled with a detectable chemical group and wherein in step(d) such detectable group is measured on the solid support as an indication of the presence of the sequence to be detected.

15. The method of claim 12 wherein the probe also comprises at least one double stranded region which, upon addition of the intercalator in step(b), forms said intercalation complexes capable of being bound by the antibody or fragment thereof.

16. The method of claim 12 wherein the liquid test medium comprises a biological sample which has been subjected to conditions to release and denature nucleic acids present therein.

17. A solid-phase hybridization method for detecting a particular polynucleotide sequence in a liquid test medium containing single stranded nucleic acids, comprising the steps of:

(a) forming a reaction mixture by contacting the liquid test medium with a nucleic acid probe, the probe comprising at least one single stranded base sequence substantially complementary to the sequence to be detected and the probe being chemically linked to a nucleic acid intercalator in the single stranded complementary region of the probe such that duplex formation in such region bearing the linked intercalator results in the formation of intercalation complexes, one of the probe and the single stranded nucleic acids from the test medium being immobilized on a solid support, such contact being performed under conditions flavorable to hybridization between the sequence to be detected and the complementary probe sequence,

(b) adding to the reaction mixture an antibody, or a fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator,

(c) separating from the remainder of the reaction mixture, the solid support carrying resulting immobilized antibody or fragment thereof bound to intercalation complexes formed between the intercalator-linked probe and the sequence to be detected, and

(d) determining the separated antibody or fragment thereof on the solid support as an indication of the presence of the sequence to be detected in the liquid test medium.

18. The method of claim 17 wherein the antibody or fragment thereof is labeled with a detectable chemical group and wherein in step(d) such detectable group is measured on the solid support as an indication of the presence of the sequence to be detected.

19. The method of claim 17 wherein the liquid test medium comprises a biological sample which has been subjected to conditions to release and denature nucleic acids present therein.

20. A solution-phase hybridization method for detecting a particular polynucleotide sequence in a liquid test medium containing single stranded nucleic acids, comprising the steps of:

(a) forming a reaction mixture by contacting the liquid test medium with a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be detected, the probe comprising a binding site for a binding substance, such contact being performed under conditions favorable to hybridization between the sequence to be detected and the complementary probe sequence,

(b) adding to the reaction mixture simultaneously or in separate steps (i) a nucleic acid intercalator, (ii) an antibody, or fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator, and (iii) an immobilized form of a binding substance for the probe,

(c) separating the resulting immobilized phase comprising antibody, or fragment thereof, bound to immobilized intercalation complexes from the remainder of the reaction mixture, and

(d) determining the separated immobilized antibody, or fragment thereof, as an indication of the presence of the sequence to be detected in the liquid test medium.

21. The method of claim 20 wherein the antibody or fragment thereof is labeled with a detectable chemical group and wherein in step(d) such detectable group is measured in the immobilized phase as an indication of the presence of the sequence to be detected.

22. The method of claim 20 wherein the probe comprises a biotin or hapten moiety and the immobilized binding substance is avidin or an anti-hapten antibody, respectively.

23. The method of claim 20 wherein the liquid test medium comprises a biological sample which has been subjected to conditions to release and denature nucleic acids present therein.

24. A test kit for detecting a particular polynucleotide sequence in a test medium, comprising in a packaged combination:

(1) a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be detected,

(2) a nucleic acid intercalator, and

(3) an antibody, or a fragment thereof, capable of binding with intercalation complexes comprising hybridized double stranded nucleic acid complexed with the intercalator.

25. The test kit of claim 24 wherein the antibody or fragment thereof is labeled with a detectable chemical group.

26. The test kit of claim 25 wherein the detectable chemical group is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.

27. The test kit of claim 25 wherein the detectable chemical group is an enzyme.

28. The test kit of claim 24 which additionally comprises a solid support for immobilizing single stranded nucleic acids from the test medium.

29. The test kit of claim 24 wherein the probe is immobilized on a solid support.

30. The test kit of claim 24 wherein the probe comprises a binding site for a binding substance and the reagent system additionally comprises an immobilized form of such binding substance.

31. The test kit of claim 30 wherein the probe comprises a biotin or hapten moiety and the immobilized binding substance is avidin or an anti-hapten antibody, respectively.

32. The test kit of claim 24 wherein the intercalator is a separate, free compound, substantially uncomplexed with nucleic acids.

33. The test kit of claim 32 wherein the probe additionally comprises at least one double stranded region.

34. The test kit of claim 24 wherein the intercalator is chemically linked to a single stranded region of the probe such that duplex formation in such region results in the formation of intercalation complexes.

35. The test kit of claim 24 for use in a sandwich hybridization format which comprises a second nucleic acid probe, the first and second probes respectively comprising at least one single stranded base sequence which is substantially complementary to a mutually exclusive portion of the sequence to be detected.

36. The test kit of claim 35 wherein one of the probes is immobilized.

37. The test kit of claim 36 wherein a single stranded region of the probe that is not immobilized is chemically linked to the intercalator such that duplex formation in such region results in the formation of intercalation complexes.

38. The test kit of claim 24 wherein the intercalator is selected from acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.

39. The test kit of claim 24 which additionally comprises a denaturation agent capable of converting double stranded nucleic acids in a test sample into single stranded form.

40. A method for detecting double stranded nucleic acid in a liquid medium, comprising the steps of:

(a) adding to said medium (i) a nucleic acid intercalator and (ii) an antibody, or a fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator, and

(b) detecting the binding of said antibody or fragment thereof to said complex.

41. The method of claim 40 wherein the antibody or fragment thereof is labeled with a detectable chemical group.

42. The method of claim 41 wherein the detectable chemical group is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.

43. The method of claim 40 wherein the intercalator is selected from acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.