Extended dynamic range assays

• US 6,180,340 B1
• Filed: 10/31/1997
• Issued: 01/30/2001
• Est. Priority Date: 10/31/1997
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Nelson, Norman C.
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US08/962,033
• Time in Patent Office: 1,187 Days
• Field of Search: 435/6, 435/7.1, 435/7.4, 435/7.5, 435/7.7, 435/7.9, 435/7.91, 435/962, 435/968, 435/7.21, 436/544-546, 436/537, 436/800, 436/814, 436/817, 436/815
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• 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 436/814 Pregnancy
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10S 436/817 Steroids or hormones

Abstract

A method and compositions for the detection and/or quantification of an analyte through the use of a plurality of labeled probes, with two or more said probes targeted to different regions of said analyte. In specific embodiments, the labels are separately distinguishable, and/or are present at different specific activities on the labels.

Claims

1. A method for detecting an analyte in a sample comprising the steps of:

providing a labeled probe reagent comprising two or more labeled probes, each of the labeled probes comprising a different, separately detectable label joined to a probe that specifically binds to a target region of an analyte, wherein each probe specifically binds to a target region that differs from that bound by another probe in the labeled probe reagent;

contacting

i) a sample containing the analyte, and

ii) the labeled probe reagent, wherein each of the labeled probes is present in a predetermined molar amount sufficient to specifically bind to a predetermined amount of the analyte, and wherein the predetermined molar amount of each labeled probe differs from that of another labeled probe present in the labeled probe reagent thereby allowing the labeled probe reagent to detect the analyte over a concentration range that is greater than the concentration range of the analyte detected by one labeled probe present in the labeled probe reagent;

incubating the sample and the labeled probe reagent under conditions favoring specific binding of the probes with the analyte, thereby forming at least one specific binding complex comprising at least one labeled probe and the analyte; and

detecting at least one separately detectable label present in a specific binding complex, thereby indicating that the analyte is present in the sample in a concentration range detected by the separately detectable label detected, wherein each separately detectable label is distinguishable from another separately detectable label.

2. The method of claim 1, wherein the incubating and detecting steps are performed in a single vessel.

3. The method of claim 1, wherein the contacting, incubating and detecting steps are performed in a single vessel.

4. The method of claim 1, wherein the labeled probe reagent of the providing step comprises labeled probes in which each different, separately detectable label is a luminescent compound.

5. The method of claim 4, wherein each different, separately detectable label is a fluorescent compound or a chemiluminescent compound.

6. The method of claim 4, wherein the labeled probe reagent comprises at least one labeled probe comprising a separately detectable label that is a fluorescent compound and at least one labeled probe comprising a separately detectable label that is a chemiluminescent compound.

7. The method of claim 4, wherein the labeled probe reagent comprises at least one labeled probe comprising a separately detectable label that is a first chemiluminescent compound and at least one labeled probe comprising a separately detectable label that is a second chemiluminescent compound.

8. The method of claim 7, wherein the first chemiluminescent compound is selected from the group consisting of standard acridinium ester (AE), naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE and 1- or 3-methyl-meta-difluoro-AE;

and

wherein the second chemiluminescent compound is selected from the group consisting of standard acridinium ester (AE), naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE and 1- or 3-methyl-meta-difluoro-AE,

provided that the first chemiluminescent compound is different from the second chemiluminescent compound.

9. The method of claim 8, wherein the first chemiluminescent compound is ortho-fluoro-AE, and wherein the second chemiluminescent compound is 2-methyl-AE.

10. The method of claim 7, wherein the labeled probe reagent further comprises at least one labeled probe comprising a separately detectable label that is a third chemiluminescent compound.

11. The method of claim 10, wherein the first chemiluminescent compound is selected from the group consisting of standard acridinium ester (AE), naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE and 1- or 3-methyl-meta-difluoro-AE;

wherein the second chemiluminescent compound is selected from the group consisting of standard acridinium ester (AE), naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE and 1- or 3-methyl-meta-difluoro-AE; and

wherein the third chemiluminescent compound is selected from the group consisting of standard acridinium ester (AE), naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE and 1- or 3-methyl-meta-difluoro-AE,

provided that the first, second and third chemiluminescent compounds differ from each other.

12. The method of claim 11, wherein the first chemiluminescent compound is 1- or 3-methyl-meta-difluoro-AE, wherein the second chemiluminescent compound is 1- or 3-methyl-AE, and wherein the third chemiluminescent compound is ortho-methoxy(cinnamyl)-AE.

13. The method of claim 1, wherein the analyte is a protein, nucleic acid, carbohydrate or lipid.

14. The method of claim 1, wherein the analyte is a nucleic acid.

15. The method of claim 1, wherein each of the labeled probes is a labeled protein or a labeled nucleic acid.

16. The method of claim 15, wherein each of the labeled probes is a labeled nucleic acid.

17. The method of claim 16, wherein each of the labeled probes is a labeled oligonucleotide.

18. The method of claim 1, wherein each of the labeled probes has a different specific activity, such that a probe having a relatively high specific activity is present in a molar amount that is lower than a molar amount of a probe having a relatively low specific activity.

19. The method of claim 1 or claim 18, wherein the providing step provides a labeled probe reagent in which each of the labeled probes is present in a molar amount that differs by at least 10-fold from that of each other labeled probe.

20. The method of claim 1 or claim 18, wherein the providing step provides a labeled probe reagent in which each of the labeled probes is present in a molar amount that differs by at least 100-fold from that of each other labeled probe.

21. The method of claim 1 or claim 18, wherein the providing step provides a labeled probe reagent in which each of the labeled probes is present in a molar amount that differs by at least 1,000-fold from that of each other labeled probe.

22. The method of claim 1 or claim 18, wherein the providing step provides a labeled probe reagent in which each of the labeled probes is present in a molar amount that differs by at least 10,000-fold from that of each other labeled probe.

23. The method of claim 18, wherein the labeled probe reagent comprises labeled probes in which each different, separately detectable label is a luminescent compound.

24. The method of claim 23, wherein each different, separately detectable label is a fluorescent compound or a chemiluminescent compound.

25. The method of claim 23, wherein the labeled probe reagent comprises at least one labeled probe comprising a separately detectable label that is a fluorescent compound and at least one labeled probe comprising a separately detectable label that is a chemiluminescent compound.

26. The method of claim 23, wherein the labeled probe reagent comprises at least one labeled probe comprising a separately detectable label that is a first chemiluminescent compound and at least one labeled probe comprising a separately detectable label that is a second chemiluminescent compound.

27. The method of claim 26, wherein the first chemiluminescent compound is selected from the group consisting of standard acridinium ester (AE), naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE and 1- or 3-methyl-meta-difluoro-AE;

and

wherein the second chemiluminescent compound is selected from the group consisting of standard acridinium ester (AE), naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE and 1- or 3-methyl-meta-difluoro-AE,

provided that the first chemiluminescent compound is different from the second chemiluminescent compound.

28. The method of claim 27, wherein the first chemiluminescent compound is ortho-fluoro-AE, and wherein the second chemiluminescent compound is 2-methyl-AE.

29. The method of claim 28, wherein the labeled probe reagent further comprises at least one labeled probe comprising a separately detectable label that is a third chemiluminescent compound.

30. The method of claim 29, wherein the first chemiluminescent compound is selected from the group consisting of standard acridinium ester (AE), naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE and 1- or 3-methyl-meta-difluoro-AE;

wherein the second chemiluminescent compound is selected from the group consisting of standard acridinium ester (AE), naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE and 1- or 3-methyl-meta-difluoro-AE; and

wherein the third chemiluminescent compound is selected from the group consisting of standard acridinium ester (AE), naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE and 1- or 3-methyl-meta-difluoro-AE,

provided that the first, second and third chemiluminescent compounds differ from each other.

31. The method of claim 30, wherein the first chemiluminescent compound is 1- or 3-methyl-meta-difluoro-AE, wherein the second chemiluminescent compound is 1- or 3-methyl-AE, and wherein the third chemiluminescent compound is ortho-methoxy(cinnamyl)-AE.

32. A method for detecting an analyte in a sample comprising the steps of:

providing a probe reagent comprising at least two labeled probes, each of the labeled probes comprising

a different target-binding nucleic acid that binds specifically to the analyte, and

a label that is separately detectable and distinguishable from a label on another labeled probe in the probe reagent,

wherein each of the labeled probes is present in a predetermined amount sufficient to specifically bind to a predetermined amount of the analyte, and wherein the predetermined amount of each labeled probe differs from that of another labeled probe in the probe reagent such that the probe reagent can detect the analyte over a concentration range greater than the concentration range of the analyte detected by a single labeled probe present in the probe reagent;

contacting a sample containing the analyte and the probe reagent;

incubating the sample and the probe reagent under conditions favoring specific binding of the analyte and the target-binding nucleic acid, thereby forming a binding complex comprising the analyte and at least one labeled probe; and

measuring a luminescent signal resulting from at least one separately detectable label present in the binding complex, thereby indicating presence of the analyte in the sample.

33. The method of claim 32, wherein the providing step provides a probe reagent in which each of the labeled probes is present in a molar amount that differs by at least 10-fold from that of each other labeled probe.

34. The method of claim 32, wherein the providing step provides a probe reagent in which each of the labeled probes is present in a molar amount that differs by at least 100-fold from that of each other labeled probe.

35. The method of claim 32, wherein the providing step provides a probe reagent in which each of the labeled probes is present in a molar amount that differs by at least 1,000-fold from that of each other labeled probe.

36. The method of claim 32, wherein the providing step provides a probe reagent in which each of the labeled probes is present in a molar amount that differs by at least 10,000-fold from that of each other labeled probe.

37. The method of claim 32, 33, 34, 35 or 36, wherein each of the labeled probes has a different specific activity, such that a probe having a relatively high specific activity is present in a lower molar amount compared to the molar amount of a probe having a relatively low specific activity.

38. The method of claim 37, wherein each of the labeled probes comprises a chemiluminescent compound label selected from the group consisting of:

standard acridinium ester (AE), naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE and 1- or 3-methyl-meta-difluoro-AE, provided that the different target-binding nucleic acids are each labeled with a different chemiluminescent compound.

39. The method of claim 32, wherein the incubating and measuring steps are performed in a single vessel.

40. The method of claim 32, wherein the contacting, incubating and measuring steps are performed in a single vessel.

41. A method for detecting an analyte in a sample comprising the steps of:

providing a probe reagent comprising two or more labeled probes, each of the labeled probes comprising a separately detectable label joined to a probe that specifically binds to a separate target region of an analyte, such that each labeled probe specifically binds to a target region that differs from that bound by another probe in the probe reagent and each separately detectable label is distinguishable from another separately detectable label in the probe reagent;

contacting

i) a sample containing the analyte, and

ii) the probe reagent, wherein each of the labeled probes is present in a predetermined molar amount sufficient to specifically bind to a predetermined amount of the analyte, and wherein the predetermined molar amount of each labeled probe differs from that of another labeled probe present in the probe reagent thereby allowing the probe reagent to detect the analyte over an extended concentration range that is greater than the concentration range of the analyte that is detected by one labeled probe present in the probe reagent;

incubating the sample and the probe reagent under conditions favoring specific binding of the labeled probes with the analyte, thereby forming at least one specific binding complex comprising at least one labeled probe and the analyte; and

detecting at least one labeled probe present in the specific binding complex, thereby indicating that the analyte is present in the sample in a concentration range detected by the probe reagent.

42. The method of claim 41, wherein the contacting and incubating steps are preformed in a single vessel.

43. The method of claim 41, wherein the sample is not serially diluted before the contacting step.

44. The method of claim 41, wherein each of the labeled probes is labeled at a different specific activity of label such that a probe having a relatively high specific activity is present in a molar amount that is lower than a molar amount of a probe having a relatively low specific activity in the probe reagent.